Modified pseudomonas exotoxin a

ABSTRACT

The invention provides a  Pseudomonas  exotoxin A (PE) comprising an amino acid sequence having a substitution of one or more B-cell and/or T-cell epitopes. The invention further provides related chimeric molecules, as well as related nucleic acids, recombinant expression vectors, host cells, populations of cells, and pharmaceutical compositions. Methods of treating or preventing cancer in a mammal, methods of inhibiting the growth of a target cell, methods of producing the PE, and methods of producing the chimeric molecule are further provided by the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of U.S. patent applicationSer. No. 14/505,590, filed Oct. 3, 2014, which claims the benefit ofU.S. Provisional Patent Application No. 61/887,418, filed Oct. 6, 2013;61/908,464, filed Nov. 25, 2013; 61/982,051, filed Apr. 21, 2014; and62/052,665, filed Sep. 19, 2014, each of which is incorporated herein byreference in its entirety.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: one 144,473 Byte ASCII (Text) file named“718352_ST25.txt,” dated Oct. 1, 2014.

BACKGROUND OF THE INVENTION

Pseudomonas exotoxin A (PE) is a bacterial toxin with cytotoxic activitythat may be effective for destroying or inhibiting the growth ofundesirable cells, e.g., cancer cells. Accordingly, PE may be useful fortreating or preventing diseases such as, e.g., cancer. However, PE maybe highly immunogenic. Accordingly, PE administration may stimulate ananti-PE immune response including, for example, the production ofanti-PE antibodies and/or T-cells, that undesirably neutralizes thecytotoxic activity of PE. Such immunogenicity may reduce the amount ofPE that can be given to the patient which may, in turn, reduce theeffectiveness of the PE for treating the disease, e.g., cancer. Thus,there is a need for improved PE.

Several deimmunized Pseudomonas exotoxins (PE) are known in art. Thedomain II deleted versions (for example, PE24) may be less immunogenicand may cause fewer side effects (such as, for example, capillary leaksyndrome and hepatotoxicity) as compared to PE38, which contains domainII. Without being bound to a particular theory, it is believed that thereduced immunogenicity and fewer side effects of PE24 could, at least inpart, be due to the reduced size of PE24, which disadvantageouslyresults in a shorter serum half life. Different furin cleavable linkersmay be employed in PE24 variants. PE immunoconjugates have mostly useddsFv fragments as targeting moieties. Such deimmunized Pseudomonasexotoxins (PE) are described in, for example, International PatentApplication Publications WO2005052006, WO2007016150, WO2007014743,WO2007031741, WO200932954, WO201132022, WO2012/154530, and WO2012/170617.

Previous immunotoxins have many disadvantages. For example,deimmunization of previous immuntoxins has been incomplete with respectto the human B-cell epitopes because immunogenic reactions stilloccurred. In addition, the deimmunization of previous immunotoxins wasaccompanied by a reduced cytotoxic potency. For example, a LO10deimmunized PE variant described in WO 2012/170617 provided a loss ofpotency of at least 40% compared to wild type (WT) PE and other PEvariants. In International Patent Application Publication WO2013/040141,Pseudomonas exotoxins with less immunogenic B-cell epitopes have beendescribed. In the PE variant LRO10, all B-cell epitopes were removed.This, however, also led to a reduction of cytotoxicity towards tumorcells.

In addition, fusion of a dsFv with domain II deleted versions of PE(PE24) have a shorter serum half life due to their reduced overall sizeas compared to dsFv fusions with PE38. The linkers of previousimmunoxins also contained T-cell epitopes and poor developability suchas, for example, a poor stability at 37° C. In addition, previousanti-mesothelin (MSLN) immunotoxins have only used mouse-derived dsFvfragments fused to PE, which may further contribute to immunogenicity.International Patent Application Publication WO 2012/154530 refers toPseudomonas exotoxin variant chimeric molecules with short flexiblelinkers which improve the cytotoxicity towards tumor cells.

BRIEF SUMMARY OF THE INVENTION

The invention relates to deimmunized Pseudomonas exotoxins and Fabfusions thereof (e.g., humanized anti-MSLN), methods for the treatmentof cancer, stabilized pharmaceutical formulations, methods for thereduction of side effects and methods for enhancing the serum half lifeand optimizing treatment schedule.

An embodiment of the invention provides a Pseudomonas exotoxin A (PE)comprising a PE amino acid sequence, wherein one or more of amino acidresidues F443, R456, L477, R494, and L552 as defined by reference to SEQID NO: 1 are, independently, substituted, wherein the PE optionally has:

(i) a further substitution of one or more amino acid residues within oneor more B cell epitopes, and the further substitution for an amino acidwithin one or more B-cell epitopes is a substitution of, independently,one or more of amino acid residues D403, D406, R412, R427, E431, R432,D461, R463, R467, R490, R505, R513, E522, R538, E548, R551, R576, Q592,and L597 as defined by reference to SEQ ID NO: 1,

(ii) a further substitution of one or more amino acid residues withinone or more T-cell epitopes,

(iii) a deletion of one or more continuous amino acid residues ofresidues 1-273 and 285-394 as defined by SEQ ID NO: 1, or

(iv) a combination of any one, two, or three of (i)-(iii).

Another embodiment of the invention provides an isolated, mutatedPseudomonas exotoxin A (PE), comprising a sequence of the followingformula:

R¹ _(n)-FCS-R² _(n)—R³ _(n)-PE functional domain III

wherein:

n=0 or 1 independently for each of R¹, R² and R³,

R¹=1 to 10 amino acid residues

FCS=a furin cleavage sequence of amino acid residues, which sequence iscleavable by furin and has an amino end and a carboxyl end,

R²=1 to 10 amino acid residues;

R³=1 or more contiguous residues of residues 365-394 of SEQ ID NO:1;and,

PE functional domain III=residues 395-613 of SEQ ID NO:1, wherein one ormore of amino acid residues F443, R456, L477, R494, and L552 as definedby reference to SEQ ID NO: 1 are, independently, substituted, whereinthe PE optionally has:

(i) a further substitution of one or more amino acid residues within oneor more B cell epitopes, and the further substitution for an amino acidwithin one or more B-cell epitopes is a substitution of, independently,one or more of amino acid residues D403, D406, R412, R427, E431, R432,D461, R463, R467, R490, R505, R513, E522, R538, E548, R551, R576, Q592,and L597 as defined by reference to SEQ ID NO: 1,

(ii) a further substitution of one or more amino acid residues withinone or more T-cell epitopes, or

(iii) both (i) and (ii).

Another embodiment of the invention provides an isolated, mutatedPseudomonas exotoxin A (PE), comprising a sequence of the followingformula:

R¹ _(n)-FCS-R² _(n)—R³ _(n)-PE functional domain III

wherein:

n=0 or 1 independently for each of R¹, R² and R³,

R¹=1 to 10 amino acid residues

FCS=a furin cleavage sequence of amino acid residues, which sequence iscleavable by furin and has an amino end and a carboxyl end,

R²=1 to 10 amino acid residues;

R³=1 or more contiguous residues of residues 365-394 of SEQ ID NO:1;and,

PE functional domain III=residues 395-613 of SEQ ID NO:1, wherein the PEincludes an arginine at position 458, as defined by reference to SEQ IDNO: 1, and

wherein the PE has:

(a) a substitution of alanine for amino acid residue R427;

(b) a substitution of alanine for amino acid residue R463;

(c) a substitution of alanine for amino acid residue R467;

(d) a substitution of alanine for amino acid residue R490;

(e) a substitution of alanine for amino acid residue R505; and

(f) a substitution of alanine for amino acid residue R538.

Additional embodiments of the invention provide related chimericmolecules, as well as related nucleic acids, recombinant expressionvectors, host cells, populations of cells, and pharmaceuticalcompositions.

Still another embodiment of the invention provides a method of treatingor preventing cancer in a mammal comprising administering to the mammalthe inventive PE, chimeric molecule, nucleic acid, recombinantexpression vector, host cell, population of cells, or pharmaceuticalcomposition, in an amount effective to treat or prevent cancer in themammal.

Another embodiment of the invention provides a method of inhibiting thegrowth of a target cell comprising contacting the cell with theinventive PE, chimeric molecule, nucleic acid, recombinant expressionvector, host cell, population of cells, or pharmaceutical composition,in an amount effective to inhibit growth of the target cell.

Additional embodiments of the invention provide methods of producing theinventive PE and methods of producing the inventive chimeric molecule.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIGS. 1A and 1B are T cell epitope heat maps showing the strongest(>20%, black squares), medium (10-20%, dark grey squares), weak (3%-10%,light grey squares) and negative (absence of response; <3%, whitesquares) responses for naïve donors (n=50) (FIG. 1A) and previouslytreated patients (n=16) (FIG. 1B). The responses are shown as apercentage of responsive spots for each donor. Responses were clusteredusing automatic sorting based on the responsiveness of the pools.

FIGS. 2A-2D are graphs showing the response of three donor samples(FIGS. 2A-2C) and one HCL patient sample (FIG. 2D) to one of 22 peptidepools, control pool (CEFT), or no peptide after stimulation with HA22(shaded bars) or LR-R494A (unshaded bars) as measured in spot-formingcells (SFCs) per 10×10⁶ cells. * indicates statistical significancebetween HA22 and LR-R494A (p<0.01).

FIGS. 3A-3C are graphs showing the response of two donor samples (FIGS.3A-3B) and one HCL patient sample (FIG. 3C) to one of 22 peptide pools,CEFT, or no peptide after stimulation with HA22 (shaded bars) orLR-R505A (unshaded bars) as measured in SFCs per 10×10⁶ cells. *indicates statistical significance (p<0.01).

FIGS. 4A-4B are graphs showing the response of two donor samples (FIGS.4A-4B) to one of 22 peptide pools, CEFT, or no peptide after stimulationwith HA22 (shaded bars) or LR-R551A (unshaded bars) as measured in SFCsper 10×10⁶ cells. * indicates statistical significance in student T test(p<0.01).

FIGS. 5A-5D are graphs showing the response of two donor samples (FIGS.5C-5D) and two mesothelioma patient samples (A-B) after stimulation withrecombinant immunotoxin (RIT) and restimulation with peptide 93 or 94with either the wild-type (WT) amino acid sequence (black bars), L552N(dark grey bars), or L552E (unshaded bars), or treatment with no peptide(light grey bars). * indicates statistical significance in student Ttest (p<0.05).

FIGS. 6A-6C are graphs showing the response of three donor samples to 22peptide pools after stimulation with either HA22-LR (WT) (shaded bars)or LR-R427A (unshaded bars) and restimulation with the appropriatepeptides as measured in SFCs per 10×10⁶ cells. * indicates statisticalsignificance (p<0.05).

FIGS. 7A-7C are graphs showing the response of two patient samples(FIGS. 7A-7B) and one donor (FIG. 7C) to one of pools 8-22 afterstimulation with either HA22-LR (WT) (shaded bars) or LR-F443A (unshadedbars) and restimulation with the appropriate peptides as measured inSFCs per 10×10⁶ cells. * indicates statistical significance (p<0.05).

FIG. 8 is a graph showing the fold change in EC50 of HA22-LR-GGS(circles), HA22 (vertical dashes), or HA22-LR-T18 (triangles) at varioustemperatures.

FIGS. 9A-9D are graphs showing the response of four donor samples afterstimulation with RIT and restimulation with no peptide, WT peptide 67,or peptide 67 with either a valine or alanine substitution at position471 as measured in SFCs per 10×10⁶ cells.

FIGS. 10A-10E are graphs showing the response of three donor samples(FIGS. 10A-10C), an HCL patient sample (FIG. 10D), and a mesotheliomapatient sample (FIG. 10E) after stimulation with peptide 67 or 68 thatcontains an alanine mutation (white bars) or histidine mutation (greybars) at position 477 or no mutation (WT) (black bars).

FIGS. 11A and 11B are graphs showing the response of samples from ameothelioma patient (FIG. 11A) and a hairy cell leukemia (HCL) patient(FIG. 11B) after stimulation with RIT and restimulation with no peptide,peptide 93, 94 or 95 with either the WT amino acid sequence (shadedbars) or L556V (unshaded bars).

FIG. 12 is a graph showing the aggregation (% Area) of HMW cFP-0170(Fab-L 010R-456A short linker; diagonally striped bars) or HMW cFP-0171(Fab-LO10R-456A elongated liker; horizontally striped bars) as measuredby size exclusion chromatograph (SEC) after incubation at 33° C.

FIG. 13 is a graph showing the aggregation (% Area) of HMW cFP-0170(Fab-LO10R-456A short linker; diamonds) or HMW cFP-0171 (Fab-LO10R-456Aelongated liker; squares) as measured by size exclusion chromatograph(SEC) after incubation at 33° C.

FIG. 14 is a graph showing the aggregation (radius in nm) of cFP-0166(Fab-LO10R short linker; diamonds) or cFP-0174 (Fab-LO10R elongatedlinker; circles) as measured by dynamic light scattering (DLS) atvarious temperatures in a range from 25° C. to 50° C.

FIG. 15 is a graph showing the aggregation (radius in nm) of cFP-0170(Fab-LO10R-456A short linker; diamonds) or cFP-0171 (Fab-LO10R-456Aelongated linker; circles) as measured by DLS at various temperatures ina range from 25° C. to 50° C.

FIG. 16 is a graph showing the aggregation (radius in nm) of cFP-0172(Fab-LO10R-456A-551A short linker; diamonds) or cFP-0173(Fab-LO10R-456A-551A elongated linker; circles) as measured by DLS atvarious temperatures in a range from 25° C. to 50° C.

FIG. 17 is a graph showing the response of patient sera clone 9H3 to(10⁷) or (10⁸) phages/well or (10⁷) or (10⁸) phages/well with antigenpre-incubation incubated with huSS1Fab-PE24LRO10 (with 458A mutation)(left diagonally striped bars), huSS1Fab-PE24LRO10R (with 458Rbackmutation) (horizontally striped bars), or huSS1Fab-PE24LRO10R-456A(with 458R backmutation and 456A mutation) (right diagonally stripedbars) as measured in optical density at 450 nm.

FIGS. 18A and 18B are graphs showing the antigenicity of the chimericmolecules SS1P (T1), SS1-dsFv-LR-LO10R (T2), SS1-dsFv-LR-LO10R456A (T3),SS1-FABLO10R (Roche 116, T4), SS1-FABLO10R456ALongLinker (Roche 171,T5), and SS1-FABLO10RLongLinker (Roche 174, T6) with respect to 20patient sera. Two representative examples (FIGS. 18A and 18B) are shown.The Y axis is relative IC50(%).

FIG. 19 is a graph showing plasma concentration (ng/ml) of chimericmolecules SS1P38 (0.4 mg/kg (diamonds)), SS1P38 (0.2 mg/kg (squares)),Fab-PE24 (0.231 mg/kg (triangles)), (dsFv-PE24 (0.16 mg/kg (x))administered to mice over a period of time (hours (h)).

FIG. 20 is a graph showing the serum half life cFP (ng/ml) of 0.3 mg/kgcFP_0205 (squares), 0.3 mg/kg SS1P (circles), or 0.3 mg/kg cFP_0205(dashed lines) over time (hours) in cyno monkeys.

FIG. 21 is a graph showing the body weight change of of mice treatedwith control (vehicle; circles); cFP 0205 3 mg/kg i.v. 3q7d (squares onsolid line); cFP 0205 2 mg/kg i.v. 3q7d (squares on larger dashed line);cFP 0205 1 mg/kg i.v. 3q7d (squares on short dashed line); cFP 0205 0.5mg/kg i.v. 3q7d (squares on dotted line) over time (days after cellinoculation).

FIGS. 22A-22F are fluorescence images of sections of lung (FIGS.22A-22B), spleen (FIGS. 22C-22D) or liver (FIGS. 22E-22F) of micetreated with labeled cFP0205 at 10× (FIGS. 22A, 22C, 22E) or 40× (FIGS.22B, 22D, 22F) magnification.

FIGS. 22G-22L are fluorescence images of sections of lung (FIGS.22G-22H), spleen (FIGS. 22I-22J) or liver (FIGS. 22K-22L) of micetreated with labeled SS1P at 10× (FIGS. 22G, 22I, 22K) or 40× (FIGS.22H, 22J, 22L) magnification.

FIG. 23 is a graph showing the tumor size of mice that were untreated(open diamonds) or treated with paclitaxel alone, (grey diamonds) RG7787(also referred to as R205 or cFP 0205) alone (black diamonds), or acombination of RG7787 and paclitaxel (black and white diamonds) overtime measured in days.

FIG. 24 is a graph showing the tumor size of mice that were untreated(vehicle) (circles) or treated with RG7787 (squares) over time measuredin days. Each data point represents the average of mean tumor volume forn=9 animals treated with RG7787 and n=8 control animals. Error bars showstandard deviations.

FIG. 25 is a graph showing the tumor size of mice that were treated withvehicle (control) (circles), RG7787 alone (squares), paclitaxel alone(triangles), or a combination of RG7787 and paclitaxel (diamonds) overtime measured in days. Error bars show standard deviations.

FIG. 26 is a graph showing the tumor size of untreated (UT) (x) mice ormice treated with R205 alone (diamonds), a combination of R205 and taxol(squares), or taxol alone (triangles) over time measured in days.

FIG. 27A is a graph showing cell viability (%) of CD22-expressing celllines treated with various concentrations of T18 or MP RIT (ng/ml).

FIG. 27B is a graph showing the relative cytotoxic activity (%) of T18or MP RIT that had been heated to one of various temperatures (° C.).

FIGS. 27C-27D are graphs showing the cytotoxic activity (IC50 (ng/ml))of MP or T18 RIT on cells from hairy cell leukemia (HCL) (FIG. 27C) orchronic lymphocytic leukemia (CLL) (FIG. 27D) patients.

FIG. 27E is a graph showing the effect of LMB-T18 on tumor size in axenograft mouse model after four injections of 5 mg/kg (squares) ofLMB-T18, three injections of 7.5 mg/kg (triangles) OF LMB-T18, orPBS-0.2% human serum albumin (circles). Arrows represent days ofinjection for all dose groups. Broken arrow indicates additionalinjection of 5 mg/ml group. (*) P>0.01 in one-way ANOVA. Error barsindicate SD.

FIG. 27F is a graph showing the % binding of MP, HA22-LR, and LMB-T18 toserum from patients with neutralizing antibodies to MP.

DETAILED DESCRIPTION OF THE INVENTION

Pseudomonas exotoxin A (“PE”) is a bacterial toxin (molecular weight 66kD) secreted by Pseudomonas aeruginosa. The native, wild-type PEsequence (SEQ ID NO: 1) is set forth in U.S. Pat. No. 5,602,095, whichis incorporated herein by reference. Native, wild-type PE includes threestructural domains that contribute to cytotoxicity. Domain Ia (aminoacids 1-252) mediates cell binding, domain II (amino acids 253-364)mediates translocation into the cytosol, and domain III (amino acids400-613) mediates ADP ribosylation of elongation factor 2. While thestructural boundary of domain III of PE is considered to start atresidue 400, it is contemplated that domain III may require a segment ofdomain Ib to retain ADP-ribosylating activity. Accordingly, functionaldomain III is defined as residues 395-613 of PE. The function of domainIb (amino acids 365-399) remains undefined. Without being bound by aparticular theory or mechanism, it is believed that the cytotoxicactivity of PE occurs through the inhibition of protein synthesis ineukaryotic cells, e.g., by the inactivation of the ADP-ribosylation ofelongation factor 2 (EF-2).

Substitutions of PE are defined herein by reference to the amino acidsequence of PE. Thus, substitutions of PE are described herein byreference to the amino acid residue present at a particular position,followed by the amino acid with which that residue has been replaced inthe particular substitution under discussion. In this regard, thepositions of the amino acid sequence of a particular embodiment of a PEare referred to herein as the positions of the amino acid sequence ofthe particular embodiment or as the positions as defined by SEQ IDNO: 1. When the positions are as defined by SEQ ID NO: 1, then theactual positions of the amino acid sequence of a particular embodimentof a PE are defined relative to the corresponding positions of SEQ IDNO: 1 and may represent different residue position numbers than theresidue position numbers of SEQ ID NO: 1. Thus, for example,substitutions refer to a replacement of an amino acid residue in theamino acid sequence of a particular embodiment of a PE corresponding tothe indicated position of the 613-amino acid sequence of SEQ ID NO: 1with the understanding that the actual positions in the respective aminoacid sequences may be different. For example, when the positions are asdefined by SEQ ID NO: 1, the term “R490” refers to the arginine normallypresent at position 490 of SEQ ID NO: 1, “R490A” indicates that thearginine normally present at position 490 of SEQ ID NO: 1 is replaced byan alanine, while “K590Q” indicates that the lysine normally present atposition 590 of SEQ ID NO: 1 has been replaced with a glutamine. In theevent of multiple substitutions at two or more positions, the two ormore substitutions may be the same or different, i.e., each amino acidresidue of the two or more amino acid residues being substituted can besubstituted with the same or different amino acid residue unlessexplicitly indicated otherwise.

The terms “Pseudomonas exotoxin” and “PE” as used herein include PE thathas been modified from the native protein to reduce or to eliminateimmunogenicity. Such modifications may include, but are not limited to,elimination of domain Ia, various amino acid deletions in domains Ib,II, and III, single amino acid substitutions and the addition of one ormore sequences at the carboxyl terminus such as DEL and REDL (SEQ ID NO:7). See Siegall et al., J Biol. Chem., 264: 14256-14261 (1989). Suchmodified PEs may be further modified to include any of the inventivesubstitution(s) for one or more amino acid residues within one or moreT-cell and/or B-cell epitopes described herein. In an embodiment, themodified PE may be a cytotoxic fragment of native, wild-type PE.Cytotoxic fragments of PE may include those which are cytotoxic with orwithout subsequent proteolytic or other processing in the target cell(e.g., as a protein or pre-protein). In a preferred embodiment, thecytotoxic fragment of PE retains at least about 20%, preferably at leastabout 40%, more preferably at least about 50%, even more preferably atleast about 75%, more preferably at least about 90%, and still morepreferably at least about 95% of the cytotoxicity of native PE. Inparticularly preferred embodiments, the cytotoxic fragment has at leastthe cytotoxicity of native PE, and preferably has increased cytotoxicityas compared to native PE.

Modified PE that reduces or eliminates immunogenicity includes, forexample, PE4E, PE40, PE38, PE25, PE38QQR, PE38KDEL, and PE35. In anembodiment, the PE may be any of PE4E, PE40, PE38, PE25, PE38QQR (inwhich PE38 has the sequence QQR added at the C-terminus), PE38KDEL (inwhich PE38 has the sequence KDEL (SEQ ID NO: 5) added at theC-terminus), PE-LR (resistance to lysosomal degradation)(also referredto as PE24), PE24-LO10, and PE35.

In an embodiment, the PE has been modified to reduce immunogenicity bydeleting domain Ia as described in in U.S. Pat. No. 4,892,827, which isincorporated herein by reference. The PE may also be modified bysubstituting certain residues of domain Ia. In an embodiment, the PE maybe PE4E, which is a substituted PE in which domain Ia is present but inwhich the basic residues of domain Ia at positions 57, 246, 247, and 249are replaced with acidic residues (e.g., glutamic acid), as disclosed inU.S. Pat. No. 5,512,658, which is incorporated herein by reference.

PE40 is a truncated derivative of PE (Pai et al., Proc. Nat'l Acad. Sci.USA, 88: 3358-62 (1991) and Kondo et al., Biol. Chem., 263: 9470-9475(1988)). PE35 is a 35 kD carboxyl-terminal fragment of PE in which aminoacid residues 1-279 have been deleted and the molecule commences with aMet at position 280 followed by amino acids 281-364 and 381-613 ofnative PE. PE35 and PE40 are disclosed, for example, in U.S. Pat. Nos.5,602,095 and 4,892,827, each of which is incorporated herein byreference. PE25 contains the 11-residue fragment from domain II and allof domain III. In some embodiments, the PE contains only domain III.

In a preferred embodiment, the PE is PE38. PE38 contains thetranslocating and ADP ribosylating domains of PE but not thecell-binding portion (Hwang J. et al., Cell, 48: 129-136 (1987)). PE38is a truncated PE pro-protein composed of amino acids 253-364 and381-613 which is activated to its cytotoxic form upon processing withina cell (see e.g., U.S. Pat. No. 5,608,039, which is incorporated hereinby reference, and Pastan et al., Biochim. Biophys. Acta, 1333: C1-C6(1997)).

In another preferred embodiment, the PE is PE-LR. PE-LR contains adeletion of domain II except for a furin cleavage sequence (FCS)corresponding to amino acid residues 274-284 of SEQ ID NO: 1(RHRQPRGWEQL (SEQ ID NO: 8)) and a deletion of amino acid residues365-394 of domain Ib. Thus, PE-LR contains amino acid residues 274-284and 395-613 of SEQ ID NO: 1. PE-LR is described in International PatentApplication Publication WO 2009/032954, which is incorporated herein byreference. The PE-LR may, optionally, additionally comprise a GGS (SEQID NO: 283) linking peptide between the FCS and amino acid residues395-613 of SEQ ID NO: 1.

As noted above, alternatively or additionally, some or all of domain Ibmay be deleted with the remaining portions joined by a bridge ordirectly by a peptide bond. Alternatively or additionally, some of theamino portion of domain II may be deleted. Alternatively oradditionally, the C-terminal end may contain the native sequence ofresidues 609-613 (REDLK) (SEQ ID NO: 6), or may contain a variation thatmay maintain the ability of the PE to translocate into the cytosol, suchas KDEL (SEQ ID NO: 5) or REDL (SEQ ID NO: 7), and repeats of thesesequences. See, e.g., U.S. Pat. Nos. 5,854,044; 5,821,238; and 5,602,095and International Patent Application Publication WO 1999/051643, whichare incorporated herein by reference. Any form of PE in whichimmunogenicity has been eliminated or reduced can be used in combinationwith any of the inventive substitution(s) for one or more amino acidresidues within one or more T-cell and/or B-cell epitopes describedherein so long as it remains capable of cytotoxicity to targeted cells,e.g., by translocation and EF-2 ribosylation in a targeted cell.

An embodiment of the invention provides a Pseudomonas exotoxin A (PE)comprising a PE amino acid sequence wherein one or more of amino acidresidues F443, R456, L477, R494, and L552 as defined by reference to SEQID NO: 1 are, independently, substituted, wherein the PE optionally has:

(i) a further substitution of one or more amino acid residues within oneor more B cell epitopes, and the further substitution for an amino acidwithin one or more B-cell epitopes is a substitution of, independently,one or more of amino acid residues D403, D406, R412, R427, E431, R432,D461, R463, R467, R490, R505, R513, E522, R538, E548, R551, R576, Q592,and L597 as defined by reference to SEQ ID NO: 1,

(ii) a further substitution of one or more amino acid residues withinone or more T-cell epitopes,

(iii) a deletion of one or more continuous amino acid residues ofresidues 1-273 and 285-394 as defined by SEQ ID NO: 1, or

(iv) a combination of any one, two, or three of (i)-(iii).

It has been discovered that amino acid residues F443, L477, R494, andL552 are located within one or more T-cell epitopes of PE. Thus, asubstitution of one or more of amino acid residues F443, L477, R494, andL552 may, advantageously, remove one or more T cell epitope(s).Accordingly, the inventive PEs may, advantageously, be less immunogenicthan an unsubstituted (e.g., wild-type) PE.

A preferred embodiment of the invention provides an isolated, mutatedPseudomonas exotoxin A (PE), comprising a sequence of the followingformula:

R¹ _(n)-FCS-R² _(n)—R³ _(n)-PE functional domain III

wherein:

n=0 or 1 independently for each of R¹, R² and R³

R¹=1 to 10 amino acid residues

FCS=a furin cleavage sequence of amino acid residues, which sequence iscleavable by furin and has an amino end and a carboxyl end,

R²=1 to 10 amino acid residues;

R³=1 or more contiguous residues of residues 365-394 of SEQ ID NO: 1;and,

PE functional domain III=residues 395-613 of SEQ ID NO: 1,

wherein one or more of amino acid residues F443, L477, R494, and L552 asdefined by reference to SEQ ID NO: 1 are, independently substituted;

and the PE comprises optionally a further substitution of an amino acidwithin one or more B-cell epitopes.

The substitution of one or more of amino acid residues F443, L477, R494,and L552 may be a substitution of any amino acid residue for one or moreof amino acid residues F443, L477, R494, and L552. In an embodiment ofthe invention, the substitution of one or more of amino acid residuesF443, L477, R494, and L552 is a substitution of, independently, alanine,glutamic acid, histidine, or asparagine in place of one or more of aminoacid residues F443, L477, R494, and L552. In an embodiment of theinvention, the substitution of L552 is a substitution of glutamic acidor asparagine in place of L552 and the substitution of L477 is asubstitution of histidine in place of L477.

In an embodiment of the invention, the substitution of one or more ofamino acid residues F443, L477, R494, and L552 is a substitution ofalanine in place of amino acid residue F443; a substitution of histidinein place of amino acid residue L477; a substitution of alanine in placeof amino acid residue R494; and a substitution of glutamic acid orasparagine in place of amino acid residue L552.

In addition to the substitution(s) for one or more amino acid residueswithin one or more PE T-cell epitopes described herein, the inventive PEmay, optionally, also include additional substitution(s) for one or moreamino acid residues within one or more B-cell epitopes of SEQ ID NO: 1.In this regard, in an embodiment of the invention, the PE has asubstitution of one or more amino acids within one or more B-cellepitopes of SEQ ID NO: 1. In a preferred embodiment of the invention,the substitution of one or more amino acid residues within one or moreB-cell epitopes of SEQ ID NO: 1 includes a substitution of alanine,glycine, serine, or glutamine for one or more amino acids within one ormore B-cell epitopes of SEQ ID NO: 1. The substitution(s) within one ormore B-cell epitopes may, advantageously, further reduce immunogenicityby the removal of one or more B-cell epitopes. The substitution(s) maybe located within any suitable PE B-cell epitope. Exemplary B-cellepitopes are disclosed in, for example, International Patent ApplicationPublications WO 2007/016150, WO 2009/032954, and WO 2011/032022, each ofwhich is incorporated herein by reference. In a preferred embodiment,the substitution of one or more amino acids within one or more B-cellepitopes of SEQ ID NO: 1 is a substitution of alanine, glycine, serine,or glutamine, independently, in place of one or more of amino acidresidues E282, E285, P290, R313, N314, P319, D324, E327, E331, Q332,D403, D406, R412, R427, E431, R432, D461, D463, R467, Y481, R490, R505,R513, L516, E522, R538, E548, R551, R576, K590, Q592, and L597, whereinthe amino acid residues E282, E285, P290, R313, N314, P319, D324, E327,E331, Q332, D403, D406, R412, R427, E431, R432, D461, D463, R467, Y481,R490, R505, R513, L516, E522, R538, E548, R551, R576, K590, Q592, andL597 are defined by reference to SEQ ID NO: 1.

In an embodiment of the invention, the further substitution of an aminoacid within one or more B-cell epitopes is a substitution of,independently, alanine, glycine, serine, or glutamine in place of one ormore of amino acid residues E282, E285, P290, R313, N314, P319, D324,E327, E331, Q332, D403, D406, R412, R427, E431, R432, D461, R463, R467,R490, R505, R513, E522, R538, E548, R551, R576, K590, Q592, and L597, asdefined by reference to SEQ ID NO: 1. Preferably, the furthersubstitution of an amino acid within one or more B-cell epitopes is asubstitution of, independently, alanine, glycine, or serine in place ofone or more of amino acid residues R427, R505, and R551. In anespecially preferred embodiment, the substitution of one or more ofamino acid residues F443, R456, L477, R494, and L552 is a substitutionof alanine in place of amino acid residue F443, a substitution ofhistidine in place of L477, a substitution of alanine in place of R494,and a substitution of glutamic acid in place of L552, and the furthersubstitution of an amino acid within one or more B-cell epitopes is: (a)a substitution of alanine for amino acid residue R427; and (b) asubstitution of alanine for amino acid residue R505, as defined byreference to SEQ ID NO: 1.

In an embodiment of the invention, any of the PEs described herein mayhave an arginine at position 458, with reference to SEQ ID NO: 1.Without being bound to a particular theory or mechanism, it is believedthat an arginine at position 458 provides enhanced cytotoxicity.

In an embodiment of the invention, the PE has an arginine residue atposition 458, as defined by reference to SEQ ID NO: 1. In a preferredembodiment, the substitution of one or more of amino acid residues F443,R456, L477, R494, and L552 is a substitution of alanine in place ofamino acid residue R456, the PE has an arginine residue at position 458,and the further substitution of an amino acid within one or more B-cellepitopes is: (a) a substitution of alanine for amino acid residue R427;(b) a substitution of alanine for amino acid residue R463; (c) asubstitution of alanine for amino acid residue R467; (d) a substitutionof alanine for amino acid residue R490; and (e) a substitution ofalanine for amino acid residue R505; (f) a substitution of alanine foramino acid residue R538; as defined by reference to SEQ ID NO: 1.

In an embodiment of the invention, the substitution of one or more ofamino acid residues F443, R456, L477, R494, and L552 is a substitutionof alanine in place of amino acid residue F443; a substitution ofalanine in place of amino acid residue R456; a substitution of histidinein place of amino acid residue L477; a substitution of alanine in placeof amino acid residue R494; and a substitution of glutamic acid in placeof amino acid residue L552, the PE has an arginine residue at position458, and the further substitution of an amino acid within one or moreB-cell epitopes is: (a) a substitution of alanine for amino acid residueR427; (b) a substitution of alanine for amino acid residue R463; (c) asubstitution of alanine for amino acid residue R467; (d) a substitutionof alanine for amino acid residue R490; (e) a substitution of alaninefor amino acid residue R505; and (f) a substitution of alanine for aminoacid residue R538; as defined by reference to SEQ ID NO: 1.

In an embodiment of the invention, the substitution of one or more ofamino acid residues F443, R456, L477, R494, and L552 is a substitutionof alanine in place of amino acid residue R456, the PE has an arginineresidue at position 458, and the further substitution of an amino acidwithin one or more B-cell epitopes is: (a) a substitution of alanine foramino acid residue R427; (b) a substitution of alanine for amino acidresidue R463; (c) a substitution of alanine for amino acid residue R467;(d) a substitution of alanine for amino acid residue R490; (e) asubstitution of alanine for amino acid residue R505; and (f) asubstitution of alanine for amino acid residue R538; as defined byreference to SEQ ID NO: 1.

In another embodiment of the invention, the substitution of one or moreof amino acid residues F443, R456, L477, R494, and L552 is asubstitution of alanine in place of amino acid residue F443; asubstitution of alanine in place of amino acid residue R456; asubstitution of histidine in place of amino acid residue L477; asubstitution of alanine in place of amino acid residue R494; and asubstitution of asparagine in place of amino acid residue L552, the PEhas an arginine residue at position 458, and the further substitution ofan amino acid within one or more B-cell epitopes is: (a) a substitutionof alanine for amino acid residue R427; (b) a substitution of alaninefor amino acid residue R463; (c) a substitution of alanine for aminoacid residue R467; (d) a substitution of alanine for amino acid residueR490; (e) a substitution of alanine for amino acid residue R505; and (f)a substitution of alanine for amino acid residue R538; as defined byreference to SEQ ID NO: 1.

In a preferred embodiment of the invention, the substitution of one ormore of amino acid residues F443, L477, R494, and L552 is a substitutionof alanine in place of amino acid residue F443; a substitution ofhistidine in place of amino acid residue L477; a substitution of alaninein place of amino acid residue R494; and a substitution of glutamic acidin place of amino acid residue L552, the PE has an arginine residue atposition 458, and the further substitution of an amino acid within oneor more B-cell epitopes is: (a) a substitution of alanine for amino acidresidue R427; (b) a substitution of alanine for amino acid residue R456;(c) a substitution of alanine for amino acid residue R463; (d) asubstitution of alanine for amino acid residue R467; (e) a substitutionof alanine for amino acid residue R490; (f) a substitution of alaninefor amino acid residue R505; and (g) a substitution of alanine for aminoacid residue R538; as defined by reference to SEQ ID NO: 1. A preferredembodiment is a PE comprising SEQ ID NO: 285 (T14-L010R+456A).

In a preferred embodiment of the invention, the substitution of one ormore of amino acid residues F443, L477, R494, and L552 is a substitutionof alanine in place of amino acid residue F443; a substitution ofhistidine in place of amino acid residue L477; a substitution of alaninein place of amino acid residue R494; and a substitution of asparagine inplace of amino acid residue L552, the PE has an arginine residue atposition 458, and the further substitution of an amino acid within oneor more B-cell epitopes is: (a) a substitution of alanine for amino acidresidue R427; (b) a substitution of alanine for amino acid residue R456;(c) a substitution of alanine for amino acid residue R463; (d) asubstitution of alanine for amino acid residue R467; (e) a substitutionof alanine for amino acid residue R490; (f) a substitution of alaninefor amino acid residue R505; and (g) a substitution of alanine for aminoacid residue R538; as defined by reference to SEQ ID NO: 1. A preferredembodiment is a PE comprising SEQ ID NO: 286 (T15-L010R+456A).

In a preferred embodiment of the invention, the substitution of one ormore of amino acid residues F443, L477, R494, and L552 is a substitutionof alanine in place of amino acid residue F443; a substitution ofhistidine in place of amino acid residue L477; a substitution of alaninein place of amino acid residue R494; and a substitution of glutamic acidin place of amino acid residue L552, the PE has an arginine residue atposition 458, and the further substitution of an amino acid within oneor more B-cell epitopes is: (a) a substitution of alanine for amino acidresidue R427; (b) a substitution of alanine for amino acid residue R463;(c) a substitution of alanine for amino acid residue R467; (d) asubstitution of alanine for amino acid residue R490; (e) a substitutionof alanine for amino acid residue R505; and (f) a substitution ofalanine for amino acid residue R538; as defined by reference to SEQ IDNO: 1. A preferred embodiment is a PE comprising SEQ ID NO: 287(T14-L010R).

In a preferred embodiment of the invention, the substitution of one ormore of amino acid residues F443, L477, R494, and L552 is a substitutionof alanine in place of amino acid residue F443; a substitution ofhistidine in place of amino acid residue L477; a substitution of alaninein place of amino acid residue R494; and a substitution of asparagine inplace of amino acid residue L552, the PE has an arginine residue atposition 458, and the further substitution of an amino acid within oneor more B-cell epitopes is: (a) a substitution of alanine for amino acidresidue R427; (b) a substitution of alanine for amino acid residue R463;(c) a substitution of alanine for amino acid residue R467; (d) asubstitution of alanine for amino acid residue R490; (e) a substitutionof alanine for amino acid residue R505; and (f) a substitution ofalanine for amino acid residue R538; as defined by reference to SEQ IDNO: 1. A preferred embodiment is a PE comprising SEQ ID NO: 288(T15-L010R).

In a preferred embodiment, the FCS is a furin cleavage sequence (FCS)corresponding to amino acid residues 274-284 of SEQ ID NO: 1(RHRQPRGWEQL (SEQ ID NO: 8)).

In a preferred embodiment,

n=1 for R¹ and R²,

R¹=a linker of the amino acid sequence of SEQ ID NO: 282 (DKTHKASGG),

R²=a linker of the amino acid sequence of SEQ ID NO: 284 (GGGGGS), and

FCS=furin cleavage sequence (FCS) corresponding to amino acid residues274-284 of SEQ ID NO: 1 (RHRQPRGWEQL (SEQ ID NO: 8)). In an especiallypreferred embodiment, n is 0 for R³.

In an embodiment of the invention, the PE has the further substitutionof an amino acid within one or more T-cell epitopes. In this regard, thePE may comprise an amino acid sequence having a further substitution ofany amino acid in place of one or more amino acid residues at positionsR421, L422, L423, A425, R427, L429, Y439, H440, F443, L444, A446, A447,1450, 463-519, R551, L552, T554, 1555, L556, and W558 as defined byreference to SEQ ID NO: 1. In an embodiment of the invention, thefurther substitution of any amino acid in place of one or more aminoacid residues at positions R421, L422, L423, A425, R427, L429, Y439,H440, F443, L444, A446, A447, 1450, 463-519, R551, L552, T554, 1555,L556, and W558 is a substitution of one or more amino acid residues atpositions R421, L422, L423, A425, R427, L429, Y439, H440, F443, L444,A446, A447, 1450, Y470, 1471, A472, P475, A476, L477, 1493, R494, N495,L498, L499, R500, V501, Y502, V503, R505, L508, P509, R551, L552, T554,1555, L556, and W558.

The substitution of one or more amino acid residues at positions R421,L422, L423, A425, R427, L429, Y439, H440, F443, L444, A446, A447, 1450,463-519, R551, L552, T554, 1555, L556, and W558 of SEQ ID NO: 1 may be asubstitution of any amino acid residue in place of an amino acid residueat any one or more of positions R421, L422, L423, A425, R427, L429,Y439, H440, F443, L444, A446, A447, 1450, 463-519, R551, L552, T554,1555, L556, and W558 of SEQ ID NO: 1. The substitution of one or moreamino acid residues at positions R421, L422, L423, A425, R427, L429,Y439, H440, F443, L444, A446, A447, 1450, 463-519, R551, L552, T554,1555, L556, and W558 of SEQ ID NO: 1 may include, e.g., a substitutionof alanine, glycine, serine, or glutamine in place of one or more aminoacid residues at position 421, 422, 423, 425, 427, 429, 439, 440, 443,444, 446, 447, 450, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472,473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486,487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500,501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514,515, 516, 517, 518, 519, 551, 552, 554, 555, 556, and 558 of SEQ IDNO: 1. In a preferred embodiment, the substitution of one or more aminoacid residues at positions R421, L422, L423, A425, R427, L429, Y439,H440, F443, L444, A446, A447, 1450, 463-519, R551, L552, T554, 1555,L556, and W558 of SEQ ID NO: 1 is a substitution of alanine, glycine,serine, or glutamine in place of one or more of amino acid residuesR421, L422, L423, A425, R427, L429, Y439, H440, F443, L444, A446, A447,I450, Y470, I471, A472, P475, A476, L477, I493, R494, N495, L498, L499,R500, V501, Y502, V503, R505, L508, P509, R551, L552, T554, 1555, L556,and W558. One or more substitutions in one or more T cell epitopeslocated at positions R421, L422, L423, A425, R427, L429, Y439, H440,F443, L444, A446, A447, I450, 463-519, R551, L552, T554, I555, L556, andW558 of PE as defined by reference to SEQ ID NO: 1 may further reduceimmunogenicity of PE. In an embodiment, the amino acid sequence does nothave a substitution of one or more amino acid residues at positions 427,467, 485, 490, 505, 513, 516, and 551.

Preferably, the PE comprises one or more substitutions that increasecytoxicity as disclosed, for example, in International PatentApplication Publication WO 2007/016150, which is incorporated herein byreference. In this regard, an embodiment of the invention provides PEwith a substitution of an amino acid within one or more B-cell epitopesof SEQ ID NO: 1 and the substitution of an amino acid within one or moreB-cell epitopes of SEQ ID NO: 1 is a substitution of valine, leucine, orisoleucine in place of amino acid residue R490, wherein the amino acidresidue R490 is defined by reference to SEQ ID NO: 1. In an embodimentof the invention, substitution of one or more amino acid residues atpositions 313, 327, 331, 332, 431, 432, 505, 516, 538, and 590 definedby reference to SEQ ID NO: 1 with alanine or glutamine may provide a PEwith an increased cytotoxicity as disclosed, for example, inInternational Patent Application Publication WO 2007/016150, which isincorporated herein by reference. Increased cytotoxic activity anddecreased immunogenicity can occur simultaneously, and are not mutuallyexclusive. Substitutions that both increase cytotoxic activity anddecrease immunogenicity, such as substitutions of R490 to glycine or,more preferably, alanine, are especially preferred.

In an embodiment of the invention, another embodiment of the inventionprovides an isolated, mutated Pseudomonas exotoxin A (PE), comprising asequence of the following formula:

R¹ _(n)-FCS-R² _(n)—R³ _(n)-PE functional domain III

wherein:

n=0 or 1 independently for each of R¹, R² and R³,

R¹=1 to 10 amino acid residues

FCS=a furin cleavage sequence of amino acid residues, which sequence iscleavable by furin and has an amino end and a carboxyl end,

R²=1 to 10 amino acid residues;

R³=1 or more contiguous residues of residues 365-394 of SEQ ID NO: 1;and,

PE functional domain III=residues 395-613 of SEQ ID NO:1, wherein one ormore of amino acid residues F443, R456, L477, R494, and L552 as definedby reference to SEQ ID NO: 1 are, independently, substituted, whereinthe PE optionally has:

(i) a further substitution of one or more amino acid residues within oneor more B cell epitopes, and the further substitution for an amino acidwithin one or more B-cell epitopes is a substitution of, independently,one or more of amino acid residues D403, D406, R412, R427, E431, R432,D461, R463, R467, R490, R505, R513, E522, R538, E548, R551, R576, Q592,and L597 as defined by reference to SEQ ID NO: 1,

(ii) a further substitution of one or more amino acid residues withinone or more T-cell epitopes, or

(iii) both (i) and (ii).

Another embodiment of the invention provides an isolated, mutatedPseudomonas exotoxin A (PE), comprising a sequence of the followingformula:

R¹ _(n)-FCS-R² _(n)—R³ _(n)-PE functional domain III

wherein:

n=0 or 1 independently for each of R¹, R² and R³,

R¹=1 to 10 amino acid residues

FCS=a furin cleavage sequence of amino acid residues, which sequence iscleavable by furin and has an amino end and a carboxyl end,

R²=1 to 10 amino acid residues;

R³=1 or more contiguous residues of residues 365-394 of SEQ ID NO: 1;and,

PE functional domain III=residues 395-613 of SEQ ID NO:1, wherein the PEincludes an arginine residue at position 458, as defined by reference toSEQ ID NO: 1, and

wherein the PE has:

(a) a substitution of alanine for amino acid residue R427;

(b) a substitution of alanine for amino acid residue R463;

(c) a substitution of alanine for amino acid residue R467;

(d) a substitution of alanine for amino acid residue R490;

(e) a substitution of alanine for amino acid residue R505; and

(f) a substitution of alanine for amino acid residue R538.

In an embodiment of the invention, n is 0 for and R² of Formula I. Inanother embodiment of the invention, n is 1 for and R². In an embodimentof the invention, when n is 0 for and R², the PE of Formula I mayfurther comprise a GGS (SEQ ID NO: 283) linking peptide between thefurin cleavage sequence (FCS) and PE functional domain III.

Without being bound by a particular theory or mechanism, it is believedthat PEs containing the FCS undergo proteolytic processing inside targetcells, thereby activating the cytotoxic activity of the toxin. The FCSof the inventive PEs may comprise any suitable furin cleavage sequenceof amino acid residues, which sequence is cleavable by furin. Exemplaryfurin cleavage sequences are described in Duckert et al., ProteinEngineering, Design & Selection, 17(1): 107-112 (2004) and InternationalPatent Application Publication WO 2009/032954, each of which isincorporated herein by reference. In an embodiment of the invention, FCScomprises residues 274-284 of SEQ ID NO: 1 (i.e., RHRQPRGWEQL (SEQ IDNO: 8)), wherein the substitution of an amino acid within one or moreB-cell epitopes of SEQ ID NO: 1 is a substitution of alanine, glycine,serine, or glutamine for amino acid residue E282 of SEQ ID NO: 1. Othersuitable FCS amino acid sequences include, but are not limited to:R—X₁—X₂—R, wherein X₁ is any naturally occurring amino acid and X₂ isany naturally occurring amino acid (SEQ ID NO: 9), RKKR (SEQ ID NO: 10),RRRR (SEQ ID NO: 11), RKAR (SEQ ID NO: 12), SRVARS (SEQ ID NO: 13),TSSRKRRFW (SEQ ID NO: 14), ASRRKARSW (SEQ ID NO: 15), RRVKKRFW (SEQ IDNO: 16), RNVVRRDW (SEQ ID NO: 17), TRAVRRRSW (SEQ ID NO: 18), RQPR (SEQID NO: 19), RHRQPRGW (SEQ ID NO: 20), RHRQPRGWE (SEQ ID NO: 21),HRQPRGWEQ (SEQ ID NO: 22), RQPRGWE (SEQ ID NO: 23), RHRSKRGWEQL (SEQ IDNO: 24), RSKR (SEQ ID NO: 25), RHRSKRGW (SEQ ID NO: 26), HRSKRGWE (SEQID NO: 27), RSKRGWEQL (SEQ ID NO: 28), HRSKRGWEQL (SEQ ID NO: 29),RHRSKR (SEQ ID NO: 30), and R—X₁—X₂—R, wherein X₁ is any naturallyoccurring amino acid and X₂ is arginine or lysine (SEQ ID NO: 4).

In still another embodiment of the invention, PE functional domain IIIcomprises residues 395-613 of SEQ ID NO: 1, wherein one or more of aminoacid residues F443, R456, L477, R494, and L552 as defined by referenceto SEQ ID NO: 1 are, independently, substituted. Amino acid residuesF443, R456, L477, R494, and L552 may be substituted as described hereinwith respect to other aspects of the invention.

In an embodiment of the invention, the FCS is represented by the formulaP4-P3-P2-P1, wherein P4 is an amino acid residue at the amino end, P1 isan amino acid residue at the carboxyl end, P1 is an arginine or a lysineresidue, and the sequence is cleavable at the carboxyl end of P1 byfurin.

In another embodiment of the invention, the FCS (i) further comprisesamino acid residues represented by P6-P5 at the amino end, (ii) furthercomprises amino acid residues represented by P1′-P2′ at the carboxylend, (iii) wherein if P1 is an arginine or a lysine residue, P2′ istryptophan, and P4 is arginine, valine or lysine, provided that if P4 isnot arginine, then P6 and P2 are basic residues, and (iv) the sequenceis cleavable at the carboxyl end of P1 by furin.

In still another embodiment of the invention, the PE functional domainIII consists of the sequence of residues 395 to 613 of SEQ ID NO: 1.

In still another embodiment of the invention, the mutated PE comprisesone or more contiguous residues of residues 365-394 of SEQ ID NO: 1between the FCS and the PE domain III.

Aspects for the development of Pseudomonas exotoxin chimeric moleculesas anti-cancer agents include their cytotoxicity towards tumor cells,their immunogenicity towards human B-cells and human T-cells, and theirthermal stability. Thermal stability may be useful for the developmentof pharmaceutical formulations or compositions.

Therefore, in one aspect of the invention, it has been discovered thatby introducing the mutation R456A instead of R458A, it may be possibleto remove all B-cell epitopes from a PE without substantially reducingtheir cytotoxicity towards tumor cells (in case no further T-cellepitopes are removed by further substitutions). Thus, for the firsttime, a PE is provided in which all B-cell epitopes have been fullyremoved and which retains its cytotoxic activity.

An embodiment of the invention provides an isolated, mutated Pseudomonasexotoxin A (PE), comprising a sequence of the following formula:

R¹ _(n)-FCS-R² _(n)—R³ _(n)-PE functional domain III

wherein:

n=0 or 1 independently for each of R¹, R² and R³

R¹=1 to 10 amino acid residues

FCS=a furin cleavage sequence of amino acid residues, which sequence iscleavable by furin and has an amino end and a carboxyl end,

R²=1 to 10 amino acid residues;

R³=1 or more contiguous residues of residues 365-394 of SEQ ID NO: 1;and,

PE functional domain III=residues 395-613 of SEQ ID NO: 1, wherein thePE includes an arginine at position 458, as defined by reference to SEQID NO: 1, and wherein the PE has:

(a) a substitution of alanine for amino acid residue R427;

(b) a substitution of alanine for amino acid residue R463;

(c) a substitution of alanine for amino acid residue R467;

(d) a substitution of alanine for amino acid residue R490;

(e) a substitution of alanine for amino acid residue R505;

(f) a substitution of alanine for amino acid residue R538; and

(g) a substitution of alanine for amino acid residue R456.

In a preferred embodiment, the FCS=a furin cleavage sequence (FCS)corresponding to amino acid residues 274-284 of SEQ ID NO: 1(RHRQPRGWEQL (SEQ ID NO: 8)).

In a preferred embodiment,

n=1 for R¹ and R²,

R¹=a linker of the amino acid sequence of SEQ ID NO: 282 (DKTHKASGG),

R²=a linker of the amino acid sequence of SEQ ID NO: 284 (GGGGGS), and

FCS=furin cleavage sequence (FCS) corresponding to amino acid residues274-284 of SEQ ID NO: 1 (RHRQPRGWEQL (SEQ ID NO: 8)). In an especiallypreferred embodiment, n is 0 for R³.

In a preferred embodiment, PE functional domain III comprises the aminoacid sequence of SEQ ID NO: 37.

An embodiment of the invention provides an isolated, mutated Pseudomonasexotoxin A (PE), comprising a sequence of the following formula:

R¹ _(n)-FCS-R²—R³-PE functional domain III

wherein:

n=0 or 1 independently for each of R¹, R² and R³

R¹=1 to 10 amino acid residues

FCS=a furin cleavage sequence of amino acid residues, which sequence iscleavable by furin and has an amino end and a carboxyl end,

R²=1 to 10 amino acid residues;

R³=1 or more contiguous residues of residues 365-394 of SEQ ID NO: 1;and,

PE functional domain III=residues 395-613 of SEQ ID NO: 1, wherein thePE includes an arginine at position 458, as defined by reference to SEQID NO: 1.

The inventive PE may be less immunogenic than an unsubstituted PE inaccordance with the invention if the immune response to the inventive PEis diminished, quantitatively or qualitatively, as compared to theimmune response to an unsubstituted PE. A quantitative decrease inimmunogenicity encompasses a decrease in the magnitude or degree of theimmune response. The magnitude or degree of immunogenicity can bemeasured on the basis of any number of known parameters, such as adecrease in the level of cytokine (e.g., antigen-specific cytokine)production (cytokine concentration), a decrease in the number oflymphocytes activated (e.g., proliferation of lymphocytes (e.g.,antigen-specific lymphocytes)) or recruited, and/or a decrease in theproduction of antibodies (antigen-specific antibodies), etc. Aqualitative decrease in immunogenicity encompasses any change in thenature of the immune response that renders the immune response lesseffective at mediating the reduction of the cytotoxic activity of thePE. Methods of measuring immunogenicity are known in the art. Forexample, measuring the types and levels of cytokines produced canmeasure immunogenicity. Alternatively or additionally, measuring thebinding of PE to antibodies (e.g., antibodies previously exposed to PE)and/or measuring the ability of the PE to induce antibodies whenadministered to a mammal (e.g., humans, mice, and/or mice in which themouse immune system is replaced with a human immune system) can measureimmunogenicity. A less immunogenic PE may be characterized by a decreasein the production of cytokines such as any one or more of IFN-γ, TNF-α,and granzyme B, and/or a reduced stimulation of a cell-mediated immuneresponse, such as a decrease in the proliferation and activation ofT-cells and/or macrophages specific for PE as compared to that obtainedwith an unsubstituted PE. Alternatively or additionally, lessimmunogenic PE may be characterized by an increase in the production ofTGF-beta and/or IL-10 as compared to that obtained with an unsubstitutedPE. In a preferred embodiment, reduced immunogenicity is characterizedby any one or more of a decrease in T cell stimulation, a decrease in Tcell proliferation, and a decrease in T cell IFNγ and/or granzyme Bsecretion. Alternatively or additionally, a less immunogenic PE may becharacterized by a decrease in the stimulation and/or activation ofB-cells specific for PE as compared to that obtained with anunsubstituted PE. For example, less immunogenic PE may be characterizedby a decrease in the differentiation of B cells into antibody-secretingplasma cells and/or memory cells as compared to that obtained with anunsubstituted PE. Reduced immunogenicity may be characterized by any oneor more of a decrease in B cell stimulation, a decrease in B cellproliferation, and a decrease in anti-PE antibody secretion. Qualitativeand quantitative diminishment of immunogenicity can occur simultaneouslyand are not mutually exclusive.

One of ordinary skill in the art will readily appreciate that theinventive PEs can be modified in any number of ways, such that thetherapeutic or prophylactic efficacy of the inventive PEs is increasedthrough the modification. For instance, the inventive PEs can beconjugated or fused either directly or indirectly through a linker to atargeting moiety. In this regard, an embodiment of the inventionprovides a chimeric molecule comprising (a) a targeting moietyconjugated or fused to (b) any of the inventive PEs described herein.The practice of conjugating compounds, e.g., inventive PEs, to targetingmoieties is known in the art. See, for instance, Wadwa et al., J. DrugTargeting, 3: 111 (1995), and U.S. Pat. No. 5,087,616. In an embodiment,any of the inventive PEs described herein may lack a targeting moiety.In an embodiment, any of the inventive PEs described herein may have anyof the targeting moieties having any of the sequences described herein.

The term “targeting moiety” as used herein, refers to any molecule oragent that specifically recognizes and binds to a cell-surface marker,such that the targeting moiety directs the delivery of the inventive PEto a population of cells on which surface the receptor is expressed.Targeting moieties include, but are not limited to, antibodies (e.g.,monoclonal antibodies), or fragments thereof, peptides, hormones, growthfactors, cytokines, and any other natural or non-natural ligands suchas, e.g., scaffold antigen binding proteins.

Scaffold antigen binding proteins are known in the art. For example,fibronectin and designed ankyrin-repeat proteins (DARPins) have beenused as alternative scaffolds for antigen-binding domains, see, e.g.,Gebauer and Skerra, Curr. Opin. Chem. Biol., 13:245-255 (2009) andStumpp et al., Drug Discov. Today, 13:695-701 (2008), both of which areincorporated herein by reference in their entirety.

In an embodiment, a scaffold antigen binding protein is selected fromthe group consisting of CTLA-4 (Evibody); lipocalin; Protein A-derivedmolecules such as the Z-domain of Protein A (Affibody, SpA), A-domain(Avimer/Maxibody); heat shock proteins such as GroEI and GroES;transferrin (trans-body); ankyrin repeat protein (DARPin); peptideaptamer; C-type lectin domain (Tetranectin); human γ-crystallin andhuman ubiquitin (affilins); PDZ domains; scorpion toxinkunitz-typedomains of human protease inhibitors; and fibronectin (adnectin), whichhas been subjected to protein engineering in order to obtain binding toa ligand other than the natural ligand.

CTLA-4 (Cytotoxic T Lymphocyte-associated Antigen 4) is a CD28-familyreceptor expressed mainly by CD4+T-cells. Its extracellular domain has avariable domain-like Ig fold. Loops corresponding to CDRs of antibodiescan be substituted with a heterologous sequence to confer differentbinding properties. CTLA-4 molecules engineered to have differentbinding specificities are also referred to as Evibodies. For furtherdetails, see J. Immunol. Methods, 248(1-2): 31-45 (2001).

Lipocalins are a family of extracellular proteins which transport smallhydrophobic molecules such as steroids, bilins, retinoids and lipids.They have a rigid beta-sheet secondary structure with a number of loopsat the open end of the conical structure which can be engineered to bindto different target antigens. Anticalins are between 160-180 amino acidsin size, and are derived from lipocalins. For further details, seeBiochim. Biophys. Acta., 1482: 337-350 (2000), U.S. Pat. No. 7,250,297B1and US20070224633.

An affibody is a scaffold derived from Protein A of Staphylococcusaureus which can be engineered to bind to an antigen. The domainincludes a three-helical bundle of approximately 58 amino acids.Libraries have been generated by the randomization of surface residues.For further details, see Protein Eng. Des. Sel., 17: 455-462 (2004) andEP1641818A1. Avimers are multidomain proteins derived from the A-domainscaffold family. The native domains of approximately 35 amino acidsadopt a defined disulphide bonded structure. Diversity is generated byshuffling the natural variation exhibited by the family of A-domains.For further details, see Nature Biotechnology, 23(12): 1556-1561 (2005)and Expert Opinion on Investigational Drugs, 16(6): 909-917 (2007).

A transferrin is a monomeric serum transport glycoprotein. Transferrinscan be engineered to bind to different target antigens by the insertionof peptide sequences in a permissive surface loop. Examples ofengineered transferrin scaffolds include the Trans-body. For furtherdetails, see J. Biol. Chem., 274: 24066-24073 (1999).

Designed Ankyrin Repeat Proteins (DARPins) are derived from Ankyrinwhich is a family of proteins that mediate attachment of integralmembrane proteins to the cytoskeleton. A single ankyrin repeat is a 33residue motif consisting of two alpha-helices and a beta-turn. They canbe engineered to bind to different target antigens by randomizingresidues in the first alpha-helix and a beta-turn of each repeat. Theirbinding interface can be increased by increasing the number of modules(a method of affinity maturation). For further details, see J. Mol.Biol., 332: 489-503 (2003); PNAS, 100(4): 1700-1705 (2003); J. Mol.Biol., 369, 1015-1028 (2007); and U.S. Patent Application Publication20040132028A1.

Fibronectin is a scaffold which can be engineered to bind to antigen.Adnectins contain a backbone of the natural amino acid sequence of the10th domain of the 15 repeating units of human fibronectin type III(FN3). Three loops at one end of the beta-sandwich can be engineered toenable an Adnectin to specifically recognize a therapeutic target ofinterest. For further details see Protein Eng. Des. Sel., 18: 435-444(2005), U.S. Patent Application Publication 20080139791, InternationalPatent Application Publication WO 2005056764 and U.S. Pat. No.6,818,418B1.

Peptide aptamers are combinatorial recognition molecules that include aconstant scaffold protein, typically thioredoxin (TrxA), which containsa constrained variable peptide loop inserted at the active site. Forfurther details, see Expert Opin. Biol. Ther., 5: 783-797 (2005).

Microbodies are derived from naturally occurring microproteins of 25-50amino acids in length which contain 3-4 cysteine bridges. Examples ofmicroproteins include KalataBI, conotoxin, and knottins. Themicroproteins have a loop which can be engineered to include up to 25amino acids without affecting the overall fold of the microprotein. Forfurther details of engineered knottin domains, see International PatentApplication Publication WO 2008098796.

Other antigen binding proteins include proteins which have been used asa scaffold to engineer different target antigen binding properties,including human gamma-crystallin and human ubiquitin (affilins), kunitztype domains of human protease inhibitors, PDZ-domains of theRas-binding protein AF-6, scorpion toxins (charybdotoxin), and C-typelectin domain (tetranectins). See Chapter 7—Non-Antibody Scaffolds fromHandbook of Therapeutic Antibodies (2007, edited by Stefan Dubel) andProtein Science, 15:14-27 (2006). Epitope binding domains of the presentinvention could be derived from any of these alternative proteindomains.

The term “antibody,” as used herein, refers to whole (also known as“intact”) antibodies or antigen binding portions thereof that retainantigen recognition and binding capability. The antibody or antigenbinding portions thereof can be a naturally-occurring antibody orantigen binding portion thereof, e.g., an antibody or antigen bindingportion thereof isolated and/or purified from a mammal, e.g., mouse,rabbit, goat, horse, chicken, hamster, human, etc. The antibody orantigen binding portion thereof can be in monomeric or polymeric form.Also, the antibody or antigen binding portion thereof can have any levelof affinity or avidity for the cell surface marker. Desirably, theantibody or antigen binding portion thereof is specific for the cellsurface marker, such that there is minimal cross-reaction with otherpeptides or proteins.

The antibody may be monoclonal or polyclonal and of any isotype, e.g.,IgM, IgG (e.g. IgG, IgG2, IgG3 or IgG4), IgD, IgA or IgE.Complementarity determining regions (CDRs) of an antibody or singlechain variable fragments (Fvs) of an antibody against a target cellsurface marker can be grafted or engineered into an antibody of choiceto confer specificity for the target cell surface marker upon thatantibody. For example, the CDRs of an antibody against a target cellsurface marker can be grafted onto a human antibody framework of a knownthree dimensional structure (see, e.g., International Patent ApplicationPublications WO 1998/045322 and WO 1987/002671; U.S. Pat. Nos.5,859,205; 5,585,089; and 4,816,567; European Patent ApplicationPublication 0173494; Jones et al., Nature, 321:522 (1986); Verhoeyen etal., Science, 239: 1534 (1988), Riechmann et al., Nature, 332:323(1988); and Winter & Milstein, Nature, 349: 293 (1991)) to form anantibody that may raise little or no immunogenic response whenadministered to a human. In a preferred embodiment, the targeting moietyis a monoclonal antibody or an antigen binding portion of the monoclonalantibody.

The antigen binding portion can be any portion that has at least oneantigen binding site, such as, e.g., the variable regions or CDRs of theintact antibody. Examples of antigen binding portions of antibodiesinclude, but are not limited to, a heavy chain, a light chain, avariable or constant region of a heavy or light chain, a single chainvariable fragment (scFv), or an Fc, Fab, Fab′, Fv, or F(ab)₂′ fragment;single domain antibodies (see, e.g., Wesolowski, Med Microbiol Immunol.,198(3): 157-74 (2009); Saerens et al., Curr. Opin. Pharmacol.,8(5):600-8 (2008); Harmsen and de Haard, Appl. Microbiol. Biotechnol.,77(1): 13-22 (2007), helix-stabilized antibodies (see, e.g., Arndt etal., J. Mol. Biol., 312: 221-228 (2001); triabodies; diabodies (EuropeanPatent Application Publication 0404097; International Patent ApplicationPublication WO 1993/011161; and Hollinger et al., Proc. Natl. Acad. Sci.USA, 90: 6444-6448 (1993)); single-chain antibody molecules (“scFvs,”see, e.g., U.S. Pat. No. 5,888,773); disulfide stabilized antibodies(“dsFvs,” see, e.g., U.S. Pat. Nos. 5,747,654 and 6,558,672), and domainantibodies (“dAbs,” see, e.g., Holt et al., Trends Biotech,21(11):484-490 (2003), Ghahroudi et al., FEBS Lett., 414:521-526 (1997),Lauwereys et al., EMBO J 17:3512-3520 (1998), Reiter et al., J. Mol.Biol. 290:685-698 (1999); and Davies and Riechmann, Biotechnology,13:475-479 (2001)).

Methods of testing antibodies or antigen binding portions thereof forthe ability to bind to any cell surface marker are known in the art andinclude any antibody-antigen binding assay, such as, for example,radioimmunoassay (MA), ELISA, Western blot, immunoprecipitation, andcompetitive inhibition assays (see, e.g., Janeway et al., infra, andU.S. Patent Application Publication 2002/0197266 A1).

Suitable methods of making antibodies are known in the art. Forinstance, standard hybridoma methods are described in, e.g., Köhler andMilstein, Eur. J. Immunol., 5, 511-519 (1976), Harlow and Lane (eds.),Antibodies: A Laboratory Manual, CSH Press (1988), and C. A. Janeway etal. (eds.), Immunobiology, 5^(th) Ed., Garland Publishing, New York,N.Y. (2001)). Alternatively, other methods, such as EBV-hybridomamethods (Haskard and Archer, J. Immunol. Methods, 74(2), 361-67 (1984),and Roder et al., Methods Enzymol., 121, 140-67 (1986)), andbacteriophage vector expression systems (see, e.g., Huse et al.,Science, 246, 1275-81 (1989)) are known in the art. Further, methods ofproducing antibodies in non-human animals are described in, e.g., U.S.Pat. Nos. 5,545,806, 5,569,825, and 5,714,352, and U.S. PatentApplication Publication 2002/0197266 A1.

Phage display also can be used to generate the antibody that may be usedin the chimeric molecules of the invention. In this regard, phagelibraries encoding antigen-binding variable (V) domains of antibodiescan be generated using standard molecular biology and recombinant DNAtechniques (see, e.g., Sambrook et al. (eds.), Molecular Cloning, ALaboratory Manual, 3^(rd) Edition, Cold Spring Harbor Laboratory Press,New York (2001)). Phage encoding a variable region with the desiredspecificity are selected for specific binding to the desired antigen,and a complete or partial antibody is reconstituted comprising theselected variable domain. Nucleic acid sequences encoding thereconstituted antibody are introduced into a suitable cell line, such asa myeloma cell used for hybridoma production, such that antibodieshaving the characteristics of monoclonal antibodies are secreted by thecell (see, e.g., Janeway et al., supra, Huse et al., supra, and U.S.Pat. No. 6,265,150).

Alternatively, antibodies can be produced by transgenic mice that aretransgenic for specific heavy and light chain immunoglobulin genes. Suchmethods are known in the art and described in, for example U.S. Pat.Nos. 5,545,806 and 5,569,825, and Janeway et al., supra.

Alternatively, the antibody can be a genetically-engineered antibody,e.g., a humanized antibody or a chimeric antibody. Humanized antibodiesadvantageously provide a lower risk of side effects and can remain inthe circulation longer. Methods for generating humanized antibodies areknown in the art and are described in detail in, for example, Janeway etal., supra, U.S. Pat. Nos. 5,225,539, 5,585,089 and 5,693,761, EuropeanPatent 0239400 B1, and United Kingdom Patent 2188638. Humanizedantibodies can also be generated using the antibody resurfacingtechnology described in, for example, U.S. Pat. No. 5,639,641 andPedersen et al., J. Mol. Biol., 235, 959-973 (1994).

The targeting moiety may specifically bind to any suitable cell surfacemarker. The choice of a particular targeting moiety and/or cell surfacemarker may be chosen depending on the particular cell population to betargeted. Cell surface markers are known in the art (see, e.g., Mufsonet al., Front. Biosci., 11:337-43 (2006); Frankel et al., Clin. CancerRes., 6:326-334 (2000); and Kreitman et al., AAPS Journal, 8(3):E532-E551 (2006)) and may be, for example, a protein or a carbohydrate.In an embodiment of the invention, the targeting moiety is a ligand thatspecifically binds to a receptor on a cell surface. Exemplary ligandsinclude, but are not limited to, vascular endothelial growth factor(VEGF), Fas, TNF-related apoptosis-inducing ligand (TRAIL), a cytokine(e.g., IL-2, IL-15, IL-4, IL-13), a lymphokine, a hormone, and a growthfactor (e.g., transforming growth factor (TGFa), neuronal growth factor,epidermal growth factor).

The cell surface marker can be, for example, a cancer antigen. The term“cancer antigen” as used herein refers to any molecule (e.g., protein,peptide, lipid, carbohydrate, etc.) solely or predominantly expressed orover-expressed by a tumor cell or cancer cell, such that the antigen isassociated with the tumor or cancer. The cancer antigen can additionallybe expressed by normal, non-tumor, or non-cancerous cells. However, insuch cases, the expression of the cancer antigen by normal, non-tumor,or non-cancerous cells is not as robust as the expression by tumor orcancer cells. In this regard, the tumor or cancer cells can over-expressthe antigen or express the antigen at a significantly higher level, ascompared to the expression of the antigen by normal, non-tumor, ornon-cancerous cells. Also, the cancer antigen can additionally beexpressed by cells of a different state of development or maturation.For instance, the cancer antigen can be additionally expressed by cellsof the embryonic or fetal stage, which cells are not normally found inan adult host. Alternatively, the cancer antigen can be additionallyexpressed by stem cells or precursor cells, which cells are not normallyfound in an adult host.

The cancer antigen can be an antigen expressed by any cell of any canceror tumor, including the cancers and tumors described herein. The cancerantigen may be a cancer antigen of only one type of cancer or tumor,such that the cancer antigen is associated with or characteristic ofonly one type of cancer or tumor. Alternatively, the cancer antigen maybe a cancer antigen (e.g., may be characteristic) of more than one typeof cancer or tumor. For example, the cancer antigen may be expressed byboth breast and prostate cancer cells and not expressed at all bynormal, non-tumor, or non-cancer cells.

Exemplary cancer antigens to which the targeting moiety may specificallybind include, but are not limited to mucin 1 (MUC1; tumor-associatedepithelial mucin), melanoma associated antigen (MAGE), preferentiallyexpressed antigen of melanoma (PRAME), carcinoembryonic antigen (CEA),prostate-specific antigen (PSA), prostate specific membrane antigen(PSMA), granulocyte-macrophage colony-stimulating factor receptor(GM-CSFR), CD56, human epidermal growth factor receptor 2 (HER2/neu)(also known as erbB-2), CD5, CD7, tyrosinase tumor antigen, tyrosinaserelated protein (TRP)1, TRP2, NY-ESO-1, telomerase, and p53. In apreferred embodiment, the cell surface marker, to which the targetingmoiety specifically binds, is selected from the group consisting ofcluster of differentiation (CD) 19, CD21, CD22, CD25, CD30, CD33 (sialicacid binding Ig-like lectin 3, myeloid cell surface antigen), CD79b,CD123 (interleukin 3 receptor alpha), transferrin receptor, EGFreceptor, mesothelin, cadherin, Lewis Y, Glypican-3, FAP (fibroblastactivation protein alpha), PSMA (prostate specific membrane antigen),CA9=CAIX (carbonic anhydrase IX), L1CAM (neural cell adhesion moleculeL1), Endosialin, HER3 (activated conformation of epidermal growth factorreceptor family member 3), Alk1/BMP9 complex (anaplastic lymphoma kinase1/bone morphogenetic protein 9), TPBG=5T4 (trophoblast glycoprotein),ROR1 (receptor tyrosine kinase-like surface antigen), HER1 (activatedconformation of epidermal growth factor receptor), and CLL1 (C-typelectin domain family 12, member A). Mesothelin is expressed in, e.g.,ovarian cancer, mesothelioma, non-small cell lung cancer, lungadenocarcinoma, fallopian tube cancer, head and neck cancer, cervicalcancer, and pancreatic cancer. CD22 is expressed in, e.g., hairy cellleukemia, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia(PLL), non-Hodgkin's lymphoma, small lymphocytic lymphoma (SLL), andacute lymphatic leukemia (ALL). CD25 is expressed in, e.g., leukemiasand lymphomas, including hairy cell leukemia and Hodgkin's lymphoma.Lewis Y antigen is expressed in, e.g., bladder cancer, breast cancer,ovarian cancer, colorectal cancer, esophageal cancer, gastric cancer,lung cancer, and pancreatic cancer. CD33 is expressed in, e.g., acutemyeloid leukemia (AML), chronic myelomonocytic leukemia (CIVIL), andmyeloproliferative disorders.

In an embodiment of the invention, the targeting moiety is an antibodythat specifically binds to a cancer antigen. Exemplary antibodies thatspecifically bind to cancer antigens include, but are not limited to,antibodies against the transferrin receptor (e.g., HB21 and variantsthereof), antibodies against CD22 (e.g., RFB4 and variants thereof),antibodies against CD25 (e.g., anti-Tac and variants thereof),antibodies against mesothelin (e.g., SS1, MORAb-009, SS, HN1, HN2, MN,MB, and variants thereof) and antibodies against Lewis Y antigen (e.g.,B3 and variants thereof). In this regard, the targeting moiety may be anantibody selected from the group consisting of B3, RFB4, SS, SS1, MN,MB, HN1, HN2, HB21, and MORAb-009, and antigen binding portions thereof.Further exemplary targeting moieties suitable for use in the inventivechimeric molecules are disclosed e.g., in U.S. Pat. No. 5,242,824(anti-transferrin receptor); U.S. Pat. No. 5,846,535 (anti-CD25); U.S.Pat. No. 5,889,157 (anti-Lewis Y); U.S. Pat. No. 5,981,726 (anti-LewisY); U.S. Pat. No. 5,990,296 (anti-Lewis Y); U.S. Pat. No. 7,081,518(anti-mesothelin); U.S. Pat. No. 7,355,012 (anti-CD22 and anti-CD25);U.S. Pat. No. 7,368,110 (anti-mesothelin); U.S. Pat. No. 7,470,775(anti-CD30); U.S. Pat. No. 7,521,054 (anti-CD25); and U.S. Pat. No.7,541,034 (anti-CD22); U.S. Patent Application Publication 2007/0189962(anti-CD22); Frankel et al., Clin. Cancer Res., 6: 326-334 (2000), andKreitman et al., AAPS Journal, 8(3): E532-E551 (2006), each of which isincorporated herein by reference. In another embodiment, the targetingmoiety may include the targeting moiety of immunotoxins known in theart. Exemplary immunotoxins include, but are not limited to, LMB-2(Anti-Tac(Fv)-PE38), BL22 and HA22 (RFB4(dsFv)-PE38), SS1P (SS1(dsFv)-PE38), HB21-PE40, and variants thereof. In a preferredembodiment, the targeting moiety is the antigen binding portion of HA22.HA22 comprises a disulfide-linked Fv anti-CD22 antibody fragmentconjugated to PE38. HA22 and variants thereof are disclosed inInternational Patent Application Publications WO 2003/027135 and WO2009/032954, which are incorporated herein by reference.

The antigen binding portion of the targeting moiety may comprise a lightchain variable region and/or a heavy chain variable region. In anembodiment of the invention, the heavy chain variable region comprises aheavy chain CDR1 region, a heavy chain CDR2 region, and a heavy chainCDR3 region. In this regard, the antigen binding domain may comprise oneor more of a heavy chain CDR1 region comprising SEQ ID NO: 49, 65, 81,97, 113, 129, 145, 161, or 177; a heavy chain CDR2 region comprising SEQID NO: 53, 69, 85, 101, 117, 133, 149, 165, or 181; and a heavy chainCDR3 region comprising SEQ ID NO: 57, 73, 89, 105, 121, 137, 153, 169,or 185. Preferably, the heavy chain comprises all of SEQ ID NOs: (a) 49,53, and 57 (anti-mesothelin heavy chain CDR1-CDR3, respectively); (b)SEQ ID NOs: 65, 69, and 73 (anti-glypican-3 gc33 heavy chain CDR1-CDR3,respectively); (c) SEQ ID NOs: 81, 85, and 89 (anti-glypican 3 ab acidicheavy chain CDR1-CDR3, respectively); (d) SEQ ID NOs: 97, 101, and 105(anti-Fap heavy chain CDR1-CDR3, respectively); (e) SEQ ID NOs: 113,117, 121 (anti-PSMA heavy chain CDR1-CDR3, respectively); (f) SEQ IDNOs: 129, 133, and 137 (anti-CAIX heavy chain CDR1-CDR3, respectively);(g) SEQ ID NOs: 145, 149, and 153 (anti-L1CAM-(1) heavy chain CDR1-CDR3,respectively); (h) SEQ ID NOs: 161, 165, 169 (anti-L1CAM (2) heavy chainCDR1-CDR3, respectively); or (i) SEQ ID NOs: 177, 181, and 185(anti-L1CAM(3) heavy chain CDR1-CDR3, respectively).

In an embodiment of the invention, the light chain variable region maycomprise a light chain CDR1 region, a light chain CDR2 region, and alight chain CDR3 region. In this regard, the antigen binding domain maycomprise one or more of a light chain CDR1 region comprising SEQ ID NO:50, 66, 82, 98, 114, 130, 146, 162, or 178; a light chain CDR2 regioncomprising SEQ ID NO: 54, 70, 86, 102, 118, 134, 150, 166, or 182; and alight chain CDR3 region comprising SEQ ID NO: 58, 74, 90, 106, 122, 138,154, 170, or 186. Preferably, the light chain comprises all of (a) SEQID NOs: 50, 54, and 58 (anti-mesothelin light chain CDR1-CDR3,respectively); (b) SEQ ID NOs: 66, 70, and 74 (anti-glypican-3 ab gc33light chain CDR1-CDR-3, respectively); (c) SEQ ID NOs: 82, 86, and 90(anti-glypican 3 ab acidic light chain CDR1-CDR3, respectively); (d) SEQID NOs: 98, 102, and 106 (anti-FAP light chain CDR1-CDR3, respectively);(e) SEQ ID NOs: 114, 118, and 122 (anti-PSMA light chain CDR1-3,respectively); (f) SEQ ID NOs: 130, 134, and 138 (anti-CAIX light chainCDR1-CDR3, respectively); (g) SEQ ID NOs: 146, 150, and 154(anti-L1CAM-(1) light chain CDR1-CDR3, respectively); (h) SEQ ID NOs:162, 166, and 170 (anti-L1CAM-(2) light chain CDR1-CDR3, respectively);or (i) SEQ ID NOs: 178, 182, and 186 (anti-L1CAM-(3) light chainCDR1-CDR3, respectively).

In an especially preferred embodiment, the antigen binding portioncomprises the CDR1, CDR2, CDR3 regions of the light chain and the CDR1,CDR2, CDR3 regions of the heavy chain. In this regard, the antigenbinding portion comprises (a) SEQ ID NOs: 49, 50, 53, 54, 57, and 58;(b) SEQ ID NOs: 65, 66, 69, 70, 73, and 74; (c) SEQ ID NOs: 81, 82, 85,86, 89, and 90; (d) SEQ ID NOs: 97, 98, 101, 102, 105, and 106; (e) SEQID NOs: 113, 114, 117, 118, 121, and 122; (f) SEQ ID NOs: 129, 130, 133,134, 137, and 138; (g) SEQ ID NOs: 145, 146, 149, 150, 153, and 154; (h)SEQ ID NOs: 161, 162, 165, 166, 169, and 170; or (i) SEQ ID NOs: 177,178, 181, 182, 185, and 186.

In an embodiment, the antigen binding portion comprises frameworkregions FR1, FR2, FR3, FR4 of the heavy chain and FR1, FR2, FR3, FR4 ofthe light chain in addition to the CDR regions described above. In thisregard, the antigen binding portion may comprise a heavy chain FR1region comprising SEQ ID NO: 47, 63, 79, 95, 111, 127, 143, 159, or 175;a heavy chain FR2 region comprising SEQ ID NO: 51, 67, 83, 99, 115, 131,147, 163, or 179; a heavy chain FR3 region comprising SEQ ID NO: 55, 71,87, 103, 119, 135, 151, 167, 183; and a heavy chain FR4 regioncomprising SEQ ID NO: 59, 75, 91, 107, 123, 139, 155, 171, or 187. Theantigen binding portion may comprise a light chain FR1 region comprisingSEQ ID NO: 48, 64, 80, 96, 112, 128, 144, 160, 176; a light chain FR2region comprising SEQ ID NO: 52, 68, 84, 100, 116, 132, 148, 164, or180; a FR3 region comprising SEQ ID NO: 56, 72, 88, 104, 120, 136, 152,168, or 184; and a FR4 region comprising SEQ ID NO: 60, 76, 92, 108,124, 140, 156, 172, or 188. Preferably, the antigen binding portioncomprises the FR1, FR2, FR3, F4, CDR1, CDR2, and CDR3 regions of thelight chain and the FR1, FR2, FR3, F4, CDR1, CDR2, and CDR3 regions ofthe heavy chain. In this regard, the antigen binding portion comprises(a) SEQ ID NOs: 47-60 (anti-mesothelin); (b) SEQ ID NOs: 63-76(anti-glypican-3 ab gc33); (c) SEQ ID NOs: 79-92 (anti-glypican 3 abacidic); (d) SEQ ID NOs: 95-108 (anti-FAP); (e) SEQ ID NOs: 111-124(anti-PSMA); (f) SEQ ID NOs: 127-140 (anti-CAIX); (g) SEQ ID NOs:143-156 (anti-L1CAM(1)); (h) SEQ ID NOs: 159-172 (anti-L1CAM(2)); or (i)SEQ ID NOs: 175-188 (anti-L1CAM(3)).

In an embodiment, the light chain variable region of the antigen bindingdomain may comprise SEQ ID NO: 46, 62, 78, 94, 110, 126, 142, 158, or174. In an embodiment, the heavy chain variable region of the antigenbinding domain may comprise SEQ ID NO: 45, 61, 77, 93, 109, 125, 141,157, or 173. In a preferred embodiment, the antigen binding domaincomprises both (a) SEQ ID NOs: 45 and 46 (anti-mesothelin); (b) SEQ IDNOs: 61 and 62 (anti-glypican-3 ab gc33); (c) SEQ ID NOs: 77 and 78(anti-gc33 acidic); (d) SEQ ID NOs: 93 and 94 (anti-FAP); (e) SEQ IDNOs: 109 and 110 (anti-PSMA); (f) SEQ ID NOs: 125 and 126 (anti-CAIX);(g) SEQ ID NOs: 141 and 142 (anti-L1CAM(1)); (h) SEQ ID NOs: 157 and 158(anti-L1CAM(2)); (i) SEQ ID NOs: 173 and 174 (anti-L1CAM(3)); or (j) SEQID NOs: 93 and 290 (anti-FAP).

Another aspect of the invention is a humanized anti-mesothelin antibodycomprising the comprising the variable heavy chain domain VH of SEQ IDNO: 45 and the variable light chain domain of SEQ ID NO: 46. Thehumanized antibody was generated by using mouse anti-mesothelin antibodySS1 as a starting material and generating specific combinations ofcertain framework regions with the CDR regions of mouse SS1 antibody.The humanized antibody advantageously provides good binding properties,stability, and developability.

In an embodiment of the invention, the targeting moiety may behumanized. In an embodiment of the invention, the targeting moiety is ahumanized SS1 or an antigen binding portion of the humanized SS1. Inthis regard, the targeting moiety may comprise a light chain comprisingSEQ ID NO: 33 (cFp-0199) and a heavy chain comprising SEQ ID NO: 38(cFp-0200). In another embodiment, the targeting moiety may comprise alight chain comprising SEQ ID NOs: 31 (cFp-0199 humanized variable lightchain domain VL) and SEQ ID NO: 32 (cFp-0199 kappa constant light chaindomain). In an embodiment, the targeting moiety may comprise a heavychain comprising SEQ ID NOs: 34 (cFp-0200 humanized variable heavy chaindomain VH) and SEQ ID NO: 35 (cFp-0200 constant heavy chain domain CH1).In an embodiment, the targeting moiety comprises all of SEQ ID NOs: 31,32, 34, and 35.

In an embodiment of the invention, the chimeric molecule comprises alinker. The term “linker” as used herein, refers to any agent ormolecule that connects the inventive PE to the targeting moiety. One ofordinary skill in the art recognizes that sites on the inventive PE,which are not necessary for the function of the inventive PE, are idealsites for attaching a linker and/or a targeting moiety, provided thatthe linker and/or targeting moiety, once attached to the inventive PE,do(es) not interfere with the function of the inventive PE, i.e.,cytotoxic activity, inhibit growth of a target cell, or to treat orprevent cancer. The linker may be capable of forming covalent bonds toboth the PE and the targeting moiety. Suitable linkers are known in theart and include, but are not limited to, straight or branched-chaincarbon linkers, heterocyclic carbon linkers, and peptide linkers. Wherethe PE and the targeting moiety are polypeptides, the linker may bejoined to the amino acids through side groups (e.g., through a disulfidelinkage to cysteine). Preferably, the linkers will be joined to thealpha carbon of the amino and carboxyl groups of the terminal aminoacids.

In another aspect of the invention, it has been discovered that byintroducing specific amino acids at both sides of the furin cleavagesequence (FCS), a more stable chimeric molecule of the Pseudomonasexotoxin with a targeting moiety has been provided. In addition, furtherpotential T-cell epitopes in the linker region have been removed, whichmay be particularly advantageous when using Fab fragments of an antibodyas a targeting moiety. These chimeric molecules provide improvedthermodynamic stability compared to chimeric molecules without theseelongated linkers and retain their cytotoxicity towards tumor cells.Accordingly, in an embodiment of the invention, the linker is anelongated linker. The elongated linker may comprise SEQ ID NO: 36.

In a preferred embodiment, R¹ _(n)-FCS-R² _(n)=a linker of the aminoacid sequence of SEQ ID NO: 36. The furin cleavage site is locatedbetween amino acid residues 15 and 16 of SEQ ID NO: 36.

An embodiment of the invention provides a chimeric molecule comprising(a) a targeting moiety conjugated or fused to (b) any of the PEsdescribed herein.

An embodiment of the invention provides an isolated chimeric moleculecomprising (a) a targeting moiety conjugated or fused to (b) a mutatedPseudomonas exotoxin A (PE), comprising a sequence of the followingformula:

R¹ _(n)-FCS-R² _(n)—R³ _(n)-PE functional domain III

wherein:

n=0 or 1 independently for each of R¹, R² and R³

R¹=1 to 10 amino acid residues

FCS=a furin cleavage sequence of amino acid residues, which sequence iscleavable by furin and has an amino end and a carboxyl end,

R²=1 to 10 amino acid residues;

R³=1 or more contiguous residues of residues 365-394 of SEQ ID NO: 1;and,

PE functional domain III=residues 395-613 of SEQ ID NO: 1, wherein thePE includes an arginine at position 458, as defined by reference to SEQID NO: 1, and wherein the PE has:

(a) a substitution of alanine for amino acid residue R427;

(b) a substitution of alanine for amino acid residue R463;

(c) a substitution of alanine for amino acid residue R467;

(d) a substitution of alanine for amino acid residue R490;

(e) a substitution of alanine for amino acid residue R505;

(f) a substitution of alanine for amino acid residue R538; and

(g) a substitution of alanine for amino acid residue R456.

In a preferred embodiment of the chimeric molecule, the FCS=furincleavage sequence (FCS) corresponding to amino acid residues 274-284 ofSEQ ID NO: 1 (RHRQPRGWEQL (SEQ ID NO: 8)).

In a preferred embodiment of the chimeric molecule,

n=1 for R¹ and R²,

R¹=a linker of the amino acid sequence of SEQ ID NO: 282 (DKTHKASGG),

R²=a linker of the amino acid sequence of SEQ ID NO: 284 (GGGGGS), and

FCS=furin cleavage sequence (FCS) corresponding to amino acid residues274-284 of SEQ ID NO: 1 (RHRQPRGWEQL (SEQ ID NO: 8)). In an especiallypreferred embodiment, n is 0 for R³.

In a preferred embodiment of the chimeric molecule, R¹ _(n)-FCS-R²_(n)=a linker of the amino acid sequence of SEQ ID NO: 36 and thetargeting moiety comprises the Fab fragment of an antibody.

In a preferred embodiment, the chimeric molecule comprises the antigenbinding portion of an anti-mesothelin antibody, a linker comprising theamino acid sequence of SEQ ID NO: 36, and a PE comprising SEQ ID NO: 37(LO10R-456A). In one preferred embodiment, the antigen binding portionis the Fab fragment of an anti-mesothelin antibody comprising thevariable heavy chain domain VH of SEQ ID NO: 45 and the variable lightchain domain of SEQ ID NO: 46. In this regard, the chimeric molecule maycomprise (a) SEQ ID NOs: 39 and 40; (b) SEQ ID NOs: 41 and 42; or (c)SEQ ID NOs: 43 and 44.

In a preferred embodiment, the chimeric molecule comprises the antigenbinding portion of an anti-glypican-3 antibody, a linker comprising theamino acid sequence of SEQ ID NO: 36, and a PE comprising SEQ ID NO: 37(LO10R-456A). In a preferred embodiment, the antigen binding portion isthe Fab fragment of an anti-glypican-3 antibody comprising the variableheavy chain domain VH of SEQ ID NO: 61 and the variable light chaindomain of SEQ ID NO: 62.

In a preferred embodiment, the chimeric molecule comprises the antigenbinding portion of an anti-glypican-3 antibody, a linker comprising theamino acid sequence of SEQ ID NO: 36, and a PE comprising SEQ ID NO: 37(L010R-456A). In a preferred embodiment, the antigen binding portion isthe Fab fragment of an anti-glypican-3 antibody comprising the variableheavy chain domain VH of SEQ ID NO: 77 and the variable light chaindomain of SEQ ID NO: 78.

In a preferred embodiment, the chimeric molecule comprises the antigenbinding portion of an anti-FAP antibody, a FCS corresponding to aminoacid residues 274-284 of SEQ ID NO: 1 (RHRQPRGWEQL (SEQ ID NO: 8)) and aPE comprising SEQ ID NO: 37 (L010R-456A). In a preferred embodiment, thechimeric molecule comprises the antigen binding portion of an anti-FAPantibody, a linker comprising the amino acid sequence of SEQ ID NO: 36,and a PE comprising SEQ ID NO: 37 (L010R-456A). In a preferredembodiment, the antigen binding portion is the Fab fragment of ananti-FAP antibody comprising the CDR1, CDR2 and CDR3 of the variableheavy chain domain VH of SEQ ID NO: 93 (SEQ ID NOs: 97, 101, and 105,respectively) and the CDR1, CDR2 and CDR3 of the variable light chaindomain of SEQ ID NO: 94 (SEQ ID NOs: 98, 102, and 106, respectively). Inone preferred embodiment, the antigen binding portion is the Fabfragment of an anti-FAP antibody comprising the variable heavy chaindomain VH of SEQ ID NO: 93 and the variable light chain domain of SEQ IDNO: 94. In another preferred embodiment, the antigen binding portion isthe Fab fragment of an anti-FAP antibody comprising the variable heavychain domain VH of SEQ ID NO: 93 and the variable light chain domain VLof SEQ ID NO: 290. In this regard, the chimeric molecule may comprise(a) SEQ ID NOs: 291 and 293; (b) SEQ ID NOs: 291 and 294; or (c) SEQ IDNOs: 292 and 294.

In a preferred embodiment, the chimeric molecule comprises the antigenbinding portion of an anti-PSMA antibody, a FCS corresponding to aminoacid residues 274-284 of SEQ ID NO: 1 (RHRQPRGWEQL (SEQ ID NO: 8)) and aPE comprising SEQ ID NO: 37 (L010R-456A). In a preferred embodiment, thechimeric molecule comprises the antigen binding portion of an anti-PSMAantibody, a linker comprising the amino acid sequence of SEQ ID NO: 36,and a PE comprising SEQ ID NO: 37 (L010R-456A). In s preferredembodiment, the antigen binding portion is the Fab fragment of ananti-PSMA antibody comprising the CDR1, CDR2 and CDR3 of the variableheavy chain domain VH of SEQ ID NO: 109 (SEQ ID NOs: 113, 117, and 121,respectively) and the CDR1, CDR2 and CDR3 of the variable light chaindomain of SEQ ID NO: 110 (SEQ ID NOs: 114, 118, and 122, respectively).

In a preferred embodiment, the chimeric molecule comprises the antigenbinding portion of an anti-CAIX antibody, a FCS corresponding to aminoacid residues 274-284 of SEQ ID NO: 1 (RHRQPRGWEQL (SEQ ID NO: 8)) and aPE comprising SEQ ID NO: 37 (L010R-456A). In a preferred embodiment, thechimeric molecule comprises the antigen binding portion of an anti-CAIXantibody, a linker comprising the amino acid sequence of SEQ ID NO: 36,and a PE comprising SEQ ID NO: 37 (L010R-456A). In a preferredembodiment, the antigen binding portion is the Fab fragment of ananti-CAIX antibody comprising the CDR1, CDR2 and CDR3 of the variableheavy chain domain VH of SEQ ID NO: 125 (SEQ ID NOs: 129, 133, and 137,respectively) and the CDR1, CDR2 and CDR3 of the variable light chaindomain of SEQ ID NO: 126 (SEQ ID NOs: 130, 134, and 138, respectively).In this regard, the chimeric molecule may comprise (a) SEQ ID NOs: 295and 297 or (b) SEQ ID NOs: 296 and 297.

In a preferred embodiment, the chimeric molecule comprises the antigenbinding portion of an anti-L1CAM antibody, a FCS corresponding to aminoacid residues 274-284 of SEQ ID NO: 1 (RHRQPRGWEQL (SEQ ID NO: 8)) and aPE comprising SEQ ID NO: 37 (L010R-456A). In a preferred embodiment, thechimeric molecule comprises the antigen binding portion of an anti-L1CAMantibody, a linker comprising the amino acid sequence of SEQ ID NO: 36,and a PE comprising SEQ ID NO: 37 (L010R-456A). In a preferredembodiment, the antigen binding portion is the Fab fragment of ananti-L1CAM antibody comprising the CDR1, CDR2 and CDR3 of the variableheavy chain domain VH of SEQ ID NO: 141 (SEQ ID NOs: 145, 149, and 153,respectively) and the CDR1, CDR2 and CDR3 of the variable light chaindomain of SEQ ID NO: 142 (SEQ ID NOs: 146, 150, and 154, respectively).In a preferred embodiment, the antigen binding portion is the Fabfragment of an anti-L1CAM antibody comprising the variable heavy chaindomain VH of SEQ ID NO: 157 and the variable light chain domain of SEQID NO: 158. In a preferred embodiment, the antigen binding portion isthe Fab fragment of an anti-L1CAM antibody comprising the variable heavychain domain VH of SEQ ID NO: 173 and the variable light chain domain ofSEQ ID NO: 174.

In a preferred embodiment, the invention provides an isolated chimericmolecule comprising (a) a targeting moiety conjugated or fused to (b) amutated Pseudomonas exotoxin A (PE), comprising a sequence of thefollowing formula:

R¹ _(n)-FCS-R² _(n)—R³ _(n)-PE functional domain III

wherein:

n=0 or 1 independently for each of R¹, R² and R³

R¹=1 to 10 amino acid residues

FCS=a furin cleavage sequence of amino acid residues, which sequence iscleavable by furin and has an amino end and a carboxyl end,

R²=1 to 10 amino acid residues;

R³=1 or more contiguous residues of residues 365-394 of SEQ ID NO: 1;and,

PE functional domain III=residues 395-613 of SEQ ID NO: 1,

wherein one or more of amino acid residues F443, L477, R494, and L552 asdefined by reference to SEQ ID NO: 1 are, independently substituted;

and the PE comprises optionally a further substitution of an amino acidwithin one or more B-cell epitopes.

It has been discovered that a substitution of histidine in place ofamino acid residue L477 leads to a strong reduction of T cell responsescompared to substitutions with other amino acids (see, for example,Example 4) while at the same time improving the cytotoxic efficacy ofthe PE compared to substitutions with other amino acids (see, forexample, Examples 3, 5 and 6). Therefore, in a preferred embodiment ofthe invention with respect to the chimeric molecule, the substitution ofone or more of amino acid residues F443, L477, R494, and L552 is asubstitution of histidine in place of amino acid residue L477. Thesubstitution of glutamic acid or asparagine in place of amino acidresidue L552 also leads to further improved toxicity while reducing Tcell responses. In a preferred embodiment of the invention with respectto the chimeric molecule, the substitution of one or more of amino acidresidues F443, L477, R494, and L552 is a substitution of histidine inplace of amino acid residue L477 and a substitution of glutamic acid orasparagine in place of amino acid residue L552. In an embodiment of theinvention with respect to the chimeric molecule, the substitution of oneor more of amino acid residues F443, L477, R494, and L552 is asubstitution of alanine in place of amino acid residue F443;substitution of histidine in place of amino acid residue L477; asubstitution of alanine in place of amino acid residue R494; and asubstitution of glutamic acid or asparagine in place of amino acidresidue L552.

In a preferred embodiment of the invention with respect to the chimericmolecule, the substitution of one or more of amino acid residues F443,L477, R494, and L552 is a substitution of alanine in place of amino acidresidue F443; a substitution of histidine in place of amino acid residueL477; a substitution of alanine in place of amino acid residue R494; anda substitution of glutamic acid in place of amino acid residue L552, thePE has an arginine residue at position 458, and the further substitutionof an amino acid within one or more B-cell epitopes is: (a) asubstitution of alanine for amino acid residue R427 and (b) asubstitution of alanine for amino acid residue R505 as defined byreference to SEQ ID NO: 1. In a preferred embodiment of the invention,the PE of the chimeric molecule comprises the amino acid sequence of SEQID NO: 289 (T18/T20).

In a preferred embodiment of the invention with respect to the chimericmolecule, the substitution of one or more of amino acid residues F443,L477, R494, and L552 is a substitution of alanine in place of amino acidresidue F443; a substitution of histidine in place of amino acid residueL477; a substitution of alanine in place of amino acid residue R494; anda substitution of glutamic acid in place of amino acid residue L552, thePE has an arginine residue at position 458, and the further substitutionof an amino acid within one or more B-cell epitopes is: (a) asubstitution of alanine for amino acid residue R427; (b) a substitutionof alanine for amino acid residue R456; (c) a substitution of alaninefor amino acid residue R463; (d) a substitution of alanine for aminoacid residue R467; (e) a substitution of alanine for amino acid residueR490; (f) a substitution of alanine for amino acid residue R505; and (g)a substitution of alanine for amino acid residue R538; as defined byreference to SEQ ID NO: 1. In a preferred embodiment, the PE of thechimeric molecule comprises the amino acid sequence of SEQ ID NO: 285(T14-L010R+456A).

In a preferred embodiment of the invention with respect to the chimericmolecule, the substitution of one or more of amino acid residues F443,L477, R494, and L552 is a substitution of alanine in place of amino acidresidue F443; a substitution of histidine in place of amino acid residueL477; a substitution of alanine in place of amino acid residue R494; anda substitution of asparagine in place of amino acid residue L552, the PEhas an arginine residue at position 458, and the further substitution ofan amino acid within one or more B-cell epitopes is: (a) a substitutionof alanine for amino acid residue R427; (b) a substitution of alaninefor amino acid residue R456; (c) a substitution of alanine for aminoacid residue R463; (d) a substitution of alanine for amino acid residueR467; (e) a substitution of alanine for amino acid residue R490; (f) asubstitution of alanine for amino acid residue R505; and (g) asubstitution of alanine for amino acid residue R538; as defined byreference to SEQ ID NO: 1. In a preferred embodiment, the PE of thechimeric molecule comprises the amino acid sequence of SEQ ID NO: 286(T15-L010R+456A).

In a preferred embodiment of the invention with respect to the chimericmolecule, the substitution of one or more of amino acid residues F443,L477, R494, and L552 is a substitution of alanine in place of amino acidresidue F443; a substitution of histidine in place of amino acid residueL477; a substitution of alanine in place of amino acid residue R494; anda substitution of glutamic acid in place of amino acid residue L552, thePE has an arginine residue at position 458, and the further substitutionof an amino acid within one or more B-cell epitopes is: (a) asubstitution of alanine for amino acid residue R427; (b) a substitutionof alanine for amino acid residue R463; (c) a substitution of alaninefor amino acid residue R467; (d) a substitution of alanine for aminoacid residue R490; (e) a substitution of alanine for amino acid residueR505; and (f) a substitution of alanine for amino acid residue R538; asdefined by reference to SEQ ID NO: 1. In a preferred embodiment, the PEof the chimeric molecule comprises the amino acid sequence of SEQ ID NO:287 (T14-L010R).

In a preferred embodiment of the invention within the chimeric molecule,the substitution of one or more of amino acid residues F443, L477, R494,and L552 is a substitution of alanine in place of amino acid residueF443; a substitution of histidine in place of amino acid residue L477; asubstitution of alanine in place of amino acid residue R494; and asubstitution of asparagine in place of amino acid residue L552, the PEhas an arginine residue at position 458, and the further substitution ofan amino acid within one or more B-cell epitopes is: (a) a substitutionof alanine for amino acid residue R427; (b) a substitution of alaninefor amino acid residue R463; (c) a substitution of alanine for aminoacid residue R467; (d) a substitution of alanine for amino acid residueR490; (e) a substitution of alanine for amino acid residue R505; and (f)a substitution of alanine for amino acid residue R538; as defined byreference to SEQ ID NO: 1. In a preferred embodiment, the PE of thechimeric molecule comprises the amino acid sequence of SEQ ID NO: 288(T15-L010R).

In a preferred embodiment with respect to the chimeric molecule, theFCS=furin cleavage sequence (FCS) corresponding to amino acid residues274-284 of SEQ ID NO: 1 (RHRQPRGWEQL (SEQ ID NO: 8)).

In a preferred embodiment with respect to the chimeric molecule,

n=1 for R¹ and R²,

R¹=a linker of the amino acid sequence of SEQ ID NO: 282 (DKTHKASGG),

R²=a linker of the amino acid sequence of SEQ ID NO: 284 (GGGGGS), and

FCS=furin cleavage sequence (FCS) corresponding to amino acid residues274-284 of SEQ ID NO: 1 (RHRQPRGWEQL (SEQ ID NO: 8)). In an especiallypreferred embodiment, n is 0 for R³.

In a preferred embodiment with respect to the chimeric molecule, R¹_(n)-FCS-R² _(n)=a linker of the amino acid sequence of SEQ ID NO: 36and the targeting moiety comprises the Fab fragment of an antibody.

In a preferred embodiment, the chimeric molecule comprises the antigenbinding portion of an anti-mesothelin antibody, a linker comprising theamino acid sequence of SEQ ID NO: 36, and a PE comprising SEQ ID NO: 285(T14-L010R+456A). In a preferred embodiment, the antigen binding portionis the Fab fragment of an anti-mesothelin antibody comprising thevariable heavy chain domain VH of SEQ ID NO: 45 and the variable lightchain domain of SEQ ID NO: 46.

In a preferred embodiment, the chimeric molecule comprises the antigenbinding portion of an anti-mesothelin antibody, a linker comprising theamino acid sequence of SEQ ID NO: 36, and a PE comprising SEQ ID NO: 286(T15-L010R+456A). In a preferred embodiment, the antigen binding portionis the Fab fragment of an anti-mesothelin antibody comprising thevariable heavy chain domain VH of SEQ ID NO: 45 and the variable lightchain domain of SEQ ID NO: 46.

In a preferred embodiment, the chimeric molecule comprises the antigenbinding portion of an anti-mesothelin antibody, a linker comprising theamino acid sequence of SEQ ID NO: 36, and a PE comprising SEQ ID NO: 287(T14-L010R). In a preferred embodiment, the antigen binding portion isthe Fab fragment of an anti-mesothelin antibody comprising the variableheavy chain domain VH of SEQ ID NO: 45 and the variable light chaindomain of SEQ ID NO: 46.

In a preferred embodiment, the chimeric molecule comprises the antigenbinding portion of an anti-mesothelin antibody, a linker comprising theamino acid sequence of SEQ ID NO: 36, and a PE comprising SEQ ID NO: 288(T15-L010R). In a preferred embodiment, the antigen binding portion isthe Fab fragment of an anti-mesothelin antibody comprising the variableheavy chain domain VH of SEQ ID NO: 45 and the variable light chaindomain of SEQ ID NO: 46.

In a preferred embodiment, the chimeric molecule comprises the antigenbinding portion of an anti-glypican-3 antibody, a furin cleavagesequence (FCS) corresponding to amino acid residues 274-284 of SEQ IDNO: 1 (RHRQPRGWEQL (SEQ ID NO: 8)) and a PE comprising SEQ ID NO: 289(T18/T20). In a preferred embodiment, the chimeric molecule comprisesthe antigen binding portion of an anti-glypican-3 antibody, a linkercomprising the amino acid sequence of SEQ ID NO: 36, and a PE comprisingSEQ ID NO: 289 (T18/T20). In a preferred embodiment, the antigen bindingportion is the Fab fragment of an anti-glypican-3 antibody comprisingthe CDR1, CDR2 and CDR3 of the variable heavy chain domain VH of SEQ IDNO: 61 (SEQ ID NOs: 65, 69, and 73, respectively) and the CDR1, CDR2 andCDR3 of the variable light chain domain of SEQ ID NO: 62 (SEQ ID NOs:66, 70, and 74, respectively). In a preferred embodiment, the antigenbinding portion is the Fab fragment of an anti-glypican-3 antibodycomprising the CDR1, CDR2 and CDR3 of the variable heavy chain domain VHof SEQ ID NO: 77 (SEQ ID NO: 81, 85, and 89, respectively) and the CDR1,CDR2 and CDR3 of the variable light chain domain of SEQ ID NO: 78 (SEQID NOs: 82, 86, and 90, respectively). In a preferred embodiment, theantigen binding portion is the Fab fragment of an anti-glypican-3antibody comprising the variable heavy chain domain VH of SEQ ID NO: 61and the variable light chain domain of SEQ ID NO: 62. In a preferredembodiment, the antigen binding portion is the Fab fragment of ananti-glypican-3 antibody comprising the variable heavy chain domain VHof SEQ ID NO: 77 and the variable light chain domain of SEQ ID NO: 78.

In a preferred embodiment, the chimeric molecule comprises the antigenbinding portion of an anti-glypican-3 antibody, a furin cleavagesequence (FCS) corresponding to amino acid residues 274-284 of SEQ IDNO: 1 (RHRQPRGWEQL (SEQ ID NO: 8)) and a PE comprising SEQ ID NO: 285(T14-LO10R+456A). In a preferred embodiment, the chimeric moleculecomprises the antigen binding portion of an anti-glypican-3 antibody, alinker comprising the amino acid sequence of SEQ ID NO: 36, and a PEcomprising SEQ ID NO: 285 (T14-L010R+456A). In a preferred embodiment,the antigen binding portion is the Fab fragment of an anti-glypican-3antibody comprising the CDR1, CDR2 and CDR3 of the variable heavy chaindomain VH of SEQ ID NO: 61 (SEQ ID NO: 65, 69, and 73, respectively) andthe CDR1, CDR2 and CDR3 of the variable light chain domain of SEQ ID NO:62 (SEQ ID NOs: 66, 70, and 74, respectively). In a preferredembodiment, the antigen binding portion is the Fab fragment of ananti-glypican-3 antibody comprising the CDR1, CDR2 and CDR3 of thevariable heavy chain domain VH of SEQ ID NO: 77 (SEQ ID NOs: 81, 85, and89, respectively) and the CDR1, CDR2 and CDR3 of the variable lightchain domain of SEQ ID NO: 78 (SEQ ID NOs: 82, 86, and 90,respectively). In a preferred embodiment, the antigen binding portion isthe Fab fragment of an anti-glypican-3 antibody comprising the variableheavy chain domain VH of SEQ ID NO: 61 and the variable light chaindomain of SEQ ID NO: 62. In a preferred embodiment, the antigen bindingportion is the Fab fragment of an anti-glypican-3 antibody comprisingthe variable heavy chain domain VH of SEQ ID NO: 77 and the variablelight chain domain of SEQ ID NO: 78.

In a preferred embodiment, the chimeric molecule comprises the antigenbinding portion of an anti-glypican-3 antibody, a furin cleavagesequence (FCS) corresponding to amino acid residues 274-284 of SEQ IDNO: 1 (RHRQPRGWEQL (SEQ ID NO: 8)) and a PE comprising SEQ ID NO: 286(T15-LO10R+456A). In a preferred embodiment, the chimeric moleculecomprises the antigen binding portion of an anti-glypican-3 antibody, alinker comprising the amino acid sequence of SEQ ID NO: 36, and a PEcomprising SEQ ID NO: 286 (T15-L010R+456A). In a preferred embodiment,the antigen binding portion is the Fab fragment of an anti-glypican-3antibody comprising the CDR1, CDR2 and CDR3 of the variable heavy chaindomain VH of SEQ ID NO: 61 (SEQ ID NO: 65, 69, and 73, respectively) andthe CDR1, CDR2 and CDR3 of the variable light chain domain of SEQ ID NO:62 (SEQ ID NOs: 66, 70, and 74, respectively). In a preferredembodiment, the antigen binding portion is the Fab fragment of ananti-glypican-3 antibody comprising the CDR1, CDR2 and CDR3 of thevariable heavy chain domain VH of SEQ ID NO: 77 (SEQ ID NOs: 81, 85, and89, respectively) and the CDR1, CDR2 and CDR3 of the variable lightchain domain of SEQ ID NO: 78 (SEQ ID NOs: 82, 86, and 90,respectively). In a preferred embodiment, the antigen binding portion isthe Fab fragment of an anti-glypican-3 antibody comprising the variableheavy chain domain VH of SEQ ID NO: 61 and the variable light chaindomain of SEQ ID NO: 62. In a preferred embodiment, the antigen bindingportion is the Fab fragment of an anti-glypican-3 antibody comprisingthe variable heavy chain domain VH of SEQ ID NO: 77 and the variablelight chain domain of SEQ ID NO: 78.

In a preferred embodiment, the chimeric molecule comprises the antigenbinding portion of an anti-glypican-3 antibody, a furin cleavagesequence (FCS) corresponding to amino acid residues 274-284 of SEQ IDNO: 1 (RHRQPRGWEQL (SEQ ID NO: 8)) and a PE comprising SEQ ID NO: 287(T14-LO10R). In a preferred embodiment, the chimeric molecule comprisesthe antigen binding portion of an anti-glypican-3 antibody, a linkercomprising the amino acid sequence of SEQ ID NO: 36, and a PE comprisingSEQ ID NO: 287 (T14-LO-1OR). In a preferred embodiment, the antigenbinding portion is the Fab fragment of an anti-glypican-3 antibodycomprising the CDR1, CDR2 and CDR3 of the variable heavy chain domain VHof SEQ ID NO: 61 (SEQ ID NOs: 65, 69, and 73, respectively) and theCDR1, CDR2 and CDR3 of the variable light chain domain of SEQ ID NO: 62(SEQ ID NOs: 66, 70, and 74, respectively). In a preferred embodiment,the antigen binding portion is the Fab fragment of an anti-glypican-3antibody comprising the CDR1, CDR2 and CDR3 of the variable heavy chaindomain VH of SEQ ID NO: 77 (SEQ ID NOs: 81, 85, and 89, respectively)and the CDR1, CDR2 and CDR3 of the variable light chain domain of SEQ IDNO: 78 (SEQ ID NOs: 82, 86, and 90, respectively). In a preferredembodiment, the antigen binding portion is the Fab fragment of ananti-glypican-3 antibody comprising the variable heavy chain domain VHof SEQ ID NO: 61 and the variable light chain domain of SEQ ID NO: 62.In a preferred embodiment, the antigen binding portion is the Fabfragment of an anti-glypican-3 antibody comprising the variable heavychain domain VH of SEQ ID NO: 77 and the variable light chain domain ofSEQ ID NO: 78.

In a preferred embodiment, the chimeric molecule comprises the antigenbinding portion of an anti-glypican-3 antibody, a furin cleavagesequence (FCS) corresponding to amino acid residues 274-284 of SEQ IDNO: 1 (RHRQPRGWEQL (SEQ ID NO: 8)) and a PE comprising SEQ ID NO: 288(T15-LO10R). In a preferred embodiment, the chimeric molecule comprisesthe antigen binding portion of an anti-glypican-3 antibody, a linkercomprising the amino acid sequence of SEQ ID NO: 36, and a PE comprisingSEQ ID NO: 288 (T15-L010R). In a preferred embodiment, the antigenbinding portion is the Fab fragment of an anti-glypican-3 antibodycomprising the CDR1, CDR2 and CDR3 of the variable heavy chain domain VHof SEQ ID NO: 61 (SEQ ID NOs: 65, 69, and 73, respectively) and theCDR1, CDR2 and CDR3 of the variable light chain domain of SEQ ID NO: 62(SEQ ID NOs: 66, 70, and 74, respectively). In a preferred embodiment,the antigen binding portion is the Fab fragment of an anti-glypican-3antibody comprising the CDR1, CDR2 and CDR3 of the variable heavy chaindomain VH of SEQ ID NO: 77 (SEQ ID NOs: 81, 85, and 89, respectively)and the CDR1, CDR2 and CDR3 of the variable light chain domain of SEQ IDNO: 78 (SEQ ID NOs: 82, 86, and 90, respectively). In a preferredembodiment, the antigen binding portion is the Fab fragment of ananti-glypican-3 antibody comprising the variable heavy chain domain VHof SEQ ID NO: 61 and the variable light chain domain of SEQ ID NO: 62.In a preferred embodiment, the antigen binding portion is the Fabfragment of an anti-glypican-3 antibody comprising the variable heavychain domain VH of SEQ ID NO: 77 and the variable light chain domain ofSEQ ID NO: 78.

In a preferred embodiment, the chimeric molecule comprises the antigenbinding portion of an anti-FAP antibody, a furin cleavage sequence (FCS)corresponding to amino acid residues 274-284 of SEQ ID NO: 1(RHRQPRGWEQL (SEQ ID NO: 8)) and a PE comprising SEQ ID NO: 289(T18/T20). In a preferred embodiment, the chimeric molecule comprisesthe antigen binding portion of an anti-FAP antibody, a linker comprisingthe amino acid sequence of SEQ ID NO: 36, and a PE comprising SEQ ID NO:289 (T18/T20). In a preferred embodiment, the antigen binding portion isthe Fab fragment of an anti-FAP antibody comprising the CDR1, CDR2 andCDR3 of the variable heavy chain domain VH of SEQ ID NO: 93 (SEQ ID NOs:97, 101, and 105, respectively) and the CDR1, CDR2 and CDR3 of thevariable light chain domain of SEQ ID NO: 94 (SEQ ID NOs: 98, 102, and106, respectively). In a preferred embodiment, the antigen bindingportion is the Fab fragment of an anti-FAP antibody comprising thevariable heavy chain domain VH of SEQ ID NO: 93 and the variable lightchain domain of SEQ ID NO: 94. In another preferred embodiment, theantigen binding portion is the Fab fragment of an anti-FAP antibodycomprising the variable heavy chain domain VH of SEQ ID NO: 93 and thevariable light chain domain of SEQ ID NO: 290.

In a preferred embodiment, the chimeric molecule comprises the antigenbinding portion of an anti-FAP antibody, a furin cleavage sequence (FCS)corresponding to amino acid residues 274-284 of SEQ ID NO: 1(RHRQPRGWEQL (SEQ ID NO: 8)) and a PE comprising SEQ ID NO: 285(T14-LO10R+456A). In a preferred embodiment, the chimeric moleculecomprises the antigen binding portion of an anti-FAP antibody, a linkercomprising the amino acid sequence of SEQ ID NO: 36, and a PE comprisingSEQ ID NO: 285 (T14-L010R+456A). In a preferred embodiment, the antigenbinding portion is the Fab fragment of an anti-FAP antibody comprisingthe CDR1, CDR2 and CDR3 of the variable heavy chain domain VH of SEQ IDNO: 93 (SEQ ID NOs: 97, 101, and 105, respectively) and the CDR1, CDR2and CDR3 of the variable light chain domain of SEQ ID NO: 94 (SEQ IDNOs: 98, 102, and 106, respectively). In a preferred embodiment, theantigen binding portion is the Fab fragment of an anti-FAP antibodycomprising the variable heavy chain domain VH of SEQ ID NO: 93 and thevariable light chain domain of SEQ ID NO: 94. In another preferredembodiment, the antigen binding portion is the Fab fragment of ananti-FAP antibody comprising the variable heavy chain domain VH of SEQID NO: 93 and the variable light chain domain of SEQ ID NO: 290.

In a preferred embodiment, the chimeric molecule comprises the antigenbinding portion of an anti-FAP antibody, a furin cleavage sequence (FCS)corresponding to amino acid residues 274-284 of SEQ ID NO: 1(RHRQPRGWEQL (SEQ ID NO: 8)) and a PE comprising SEQ ID NO: 286(T15-LO10R+456A). In a preferred embodiment, the chimeric moleculecomprises the antigen binding portion of an anti-FAP antibody, a linkercomprising the amino acid sequence of SEQ ID NO: 36, and a PE comprisingSEQ ID NO: 286 (T15-L010R+456A). In a preferred embodiment, the antigenbinding portion is the Fab fragment of an anti-FAP antibody comprisingthe CDR1, CDR2 and CDR3 of the variable heavy chain domain VH of SEQ IDNO: 93 (SEQ ID NOs: 97, 101, and 105, respectively) and the CDR1, CDR2and CDR3 of the variable light chain domain of SEQ ID NO: 94 (SEQ IDNOs: 98, 102, and 106, respectively). In a preferred embodiment, theantigen binding portion is the Fab fragment of an anti-FAP antibodycomprising the variable heavy chain domain VH of SEQ ID NO: 93 and thevariable light chain domain of SEQ ID NO: 94. In another preferredembodiment, the antigen binding portion is the Fab fragment of ananti-FAP antibody comprising the variable heavy chain domain VH of SEQID NO: 93 and the variable light chain domain of SEQ ID NO: 290.

In a preferred embodiment, the chimeric molecule comprises the antigenbinding portion of an anti-FAP antibody, a furin cleavage sequence (FCS)corresponding to amino acid residues 274-284 of SEQ ID NO: 1(RHRQPRGWEQL (SEQ ID NO: 8)) and a PE comprising SEQ ID NO: 287(T14-LO10R). In a preferred embodiment, the chimeric molecule comprisesthe antigen binding portion of an anti-FAP antibody, a linker comprisingthe amino acid sequence of SEQ ID NO: 36, and a PE comprising SEQ ID NO:287 (T14-L010R). In a preferred embodiment, the antigen binding portionis the Fab fragment of an anti-FAP antibody comprising the CDR1, CDR2and CDR3 of the variable heavy chain domain VH of SEQ ID NO: 93 (SEQ IDNOs: 97, 101, and 105, respectively) and the CDR1, CDR2 and CDR3 of thevariable light chain domain of SEQ ID NO: 94 (SEQ ID NOs: 98, 102, and106,). In a preferred embodiment, the antigen binding portion is the Fabfragment of an anti-FAP antibody comprising the variable heavy chaindomain VH of SEQ ID NO: 93 and the variable light chain domain of SEQ IDNO: 94. In another preferred embodiment, the antigen binding portion isthe Fab fragment of an anti-FAP antibody comprising the variable heavychain domain VH of SEQ ID NO: 93 and the variable light chain domain ofSEQ ID NO: 290.

In a preferred embodiment, the chimeric molecule comprises the antigenbinding portion of an anti-FAP antibody, a furin cleavage sequence (FCS)corresponding to amino acid residues 274-284 of SEQ ID NO: 1(RHRQPRGWEQL (SEQ ID NO: 8)) and a PE comprising SEQ ID NO: 288(T15-LO10R). In a preferred embodiment, the chimeric molecule comprisesthe antigen binding portion of an anti-FAP antibody, a linker comprisingthe amino acid sequence of SEQ ID NO: 36, and a PE comprising SEQ ID NO:288 (T15-L010R). In a preferred embodiment, the antigen binding portionis the Fab fragment of an anti-FAP antibody comprising the CDR1, CDR2and CDR3 of the variable heavy chain domain VH of SEQ ID NO: 93 (SEQ IDNOs: 97, 101, and 105, respectively) and the CDR1, CDR2 and CDR3 of thevariable light chain domain of SEQ ID NO: 94 (SEQ ID NOs: 98, 102, and106, respectively). In a preferred embodiment, the antigen bindingportion is the Fab fragment of an anti-FAP antibody comprising thevariable heavy chain domain VH of SEQ ID NO: 93 and the variable lightchain domain of SEQ ID NO: 94. In another preferred embodiment, theantigen binding portion is the Fab fragment of an anti-FAP antibodycomprising the variable heavy chain domain VH of SEQ ID NO: 93 and thevariable light chain domain of SEQ ID NO: 290. In a preferredembodiment, the chimeric molecule comprises the antigen binding portionof an anti-PSMA antibody, a furin cleavage sequence (FCS) correspondingto amino acid residues 274-284 of SEQ ID NO: 1 (RHRQPRGWEQL (SEQ ID NO:8)) and a PE comprising SEQ ID NO: 289 (T18/T20). In a preferredembodiment, the chimeric molecule comprises the antigen binding portionof an anti-PSMA antibody, a linker comprising the amino acid sequence ofSEQ ID NO: 36, and a PE comprising SEQ ID NO: 289 (T18/T20). In apreferred embodiment, the antigen binding portion is the Fab fragment ofan anti-PSMA antibody comprising the CDR1, CDR2 and CDR3 of the variableheavy chain domain VH of SEQ ID NO: 109 (SEQ ID NOs: 113, 117, and 121,respectively) and the CDR1, CDR2 and CDR3 of the variable light chaindomain of SEQ ID NO: 110 (SEQ ID NOs: 114, 118, and 122, respectively).

In a preferred embodiment, the chimeric molecule comprises the antigenbinding portion of an anti-PSMA antibody, a furin cleavage sequence(FCS) corresponding to amino acid residues 274-284 of SEQ ID NO: 1(RHRQPRGWEQL (SEQ ID NO: 8)) and a PE comprising SEQ ID NO: 285(T14-LO10R+456A). In a preferred embodiment, the chimeric moleculecomprises the antigen binding portion of an anti-PSMA antibody, a linkercomprising the amino acid sequence of SEQ ID NO: 36, and a PE comprisingSEQ ID NO: 285 (T14-L010R+456A). In a preferred embodiment, the antigenbinding portion is the Fab fragment of an anti-PSMA antibody comprisingthe CDR1, CDR2 and CDR3 of the variable heavy chain domain VH of SEQ IDNO: 109 (SEQ ID NOs: 113, 117, and 121, respectively) and the CDR1, CDR2and CDR3 of the variable light chain domain of SEQ ID NO: 110 (SEQ IDNOs: 114, 118, and 122, respectively).

In a preferred embodiment, the chimeric molecule comprises the antigenbinding portion of an anti-PSMA antibody, a furin cleavage sequence(FCS) corresponding to amino acid residues 274-284 of SEQ ID NO: 1(RHRQPRGWEQL (SEQ ID NO: 8)) and a PE comprising SEQ ID NO: 286(T15-LO10R+456A). In a preferred embodiment, the chimeric moleculecomprises the antigen binding portion of an anti-PSMA antibody, a linkercomprising the amino acid sequence of SEQ ID NO: 36, and a PE comprisingSEQ ID NO: 286 (T15-L010R+456A). In a preferred embodiment, the antigenbinding portion is the Fab fragment of an anti-PSMA antibody comprisingthe CDR1, CDR2 and CDR3 of the variable heavy chain domain VH of SEQ IDNO: 109 (SEQ ID NOs: 113, 117, and 121, respectively) and the CDR1, CDR2and CDR3 of the variable light chain domain of SEQ ID NO: 110 (SEQ IDNOs: 114, 118, and 122, respectively).

In a preferred embodiment, the chimeric molecule comprises the antigenbinding portion of an anti-PSMA antibody, a furin cleavage sequence(FCS) corresponding to amino acid residues 274-284 of SEQ ID NO: 1(RHRQPRGWEQL (SEQ ID NO: 8)) and a PE comprising SEQ ID NO: 287(T14-LO10R). In a preferred embodiment, the chimeric molecule comprisesthe antigen binding portion of an anti-PSMA antibody, a linkercomprising the amino acid sequence of SEQ ID NO: 36, and a PE comprisingSEQ ID NO: 287 (T14-L010R). In a preferred embodiment, the antigenbinding portion is the Fab fragment of an anti-PSMA antibody comprisingthe CDR1, CDR2 and CDR3 of the variable heavy chain domain VH of SEQ IDNO: 109 (SEQ ID NOs: 113, 117, and 121, respectively) and the CDR1, CDR2and CDR3 of the variable light chain domain of SEQ ID NO: 110 (SEQ IDNOs: 114, 118, and 122, respectively).

In a preferred embodiment, the chimeric molecule comprises the antigenbinding portion of an anti-PSMA antibody, a furin cleavage sequence(FCS) corresponding to amino acid residues 274-284 of SEQ ID NO: 1(RHRQPRGWEQL (SEQ ID NO: 8)) and a PE comprising SEQ ID NO: 288(T15-LO10R). In a preferred embodiment, the chimeric molecule comprisesthe antigen binding portion of an anti-PSMA antibody, a linkercomprising the amino acid sequence of SEQ ID NO: 36, and a PE comprisingSEQ ID NO: 288 (T15-L010R). In a preferred embodiment, the antigenbinding portion is the Fab fragment of an anti-PSMA antibody comprisingthe CDR1, CDR2 and CDR3 of the variable heavy chain domain VH of SEQ IDNO: 109 (SEQ ID NOs: 113, 117, and 121, respectively) and the CDR1, CDR2and CDR3 of the variable light chain domain of SEQ ID NO: 110 (SEQ IDNOs: 114, 118, and 122, respectively).

In a preferred embodiment, the chimeric molecule comprises the antigenbinding portion of an anti-CAIX antibody, a furin cleavage sequence(FCS) corresponding to amino acid residues 274-284 of SEQ ID NO: 1(RHRQPRGWEQL (SEQ ID NO: 8)) and a PE comprising SEQ ID NO: 289(T18/T20). In a preferred embodiment, the chimeric molecule comprisesthe antigen binding portion of an anti-CAIX antibody, a linkercomprising the amino acid sequence of SEQ ID NO: 36, and a PE comprisingSEQ ID NO: 289 (T18/T20). In a preferred embodiment, the antigen bindingportion is the Fab fragment of an anti-CAIX antibody comprising theCDR1, CDR2 and CDR3 of the variable heavy chain domain VH of SEQ ID NO:125 (SEQ ID NOs: 129, 133, and 137, respectively) and the CDR1, CDR2 andCDR3 of the variable light chain domain of SEQ ID NO: 126 (SEQ ID NOs:130, 134, and 138,).

In a preferred embodiment, the chimeric molecule comprises the antigenbinding portion of an anti-CAIX antibody, a furin cleavage sequence(FCS) corresponding to amino acid residues 274-284 of SEQ ID NO: 1(RHRQPRGWEQL (SEQ ID NO: 8)) and a PE comprising SEQ ID NO: 285(T14-LO10R+456A). In a preferred embodiment, the chimeric moleculecomprises the antigen binding portion of an anti-CAIX antibody, a linkercomprising the amino acid sequence of SEQ ID NO: 36, and a PE comprisingSEQ ID NO: 285 (T14-L010R+456A). In a preferred embodiment, the antigenbinding portion is the Fab fragment of an anti-CAIX antibody comprisingthe CDR1, CDR2 and CDR3 of the variable heavy chain domain VH of SEQ IDNO: 125 (SEQ ID NOs: 129, 133, and 137, respectively) and the CDR1, CDR2and CDR3 of the variable light chain domain of SEQ ID NO: 126 (SEQ IDNOs: 130, 134, and 138, respectively).

In a preferred embodiment, the chimeric molecule comprises the antigenbinding portion of an anti-CAIX antibody, a furin cleavage sequence(FCS) corresponding to amino acid residues 274-284 of SEQ ID NO: 1(RHRQPRGWEQL (SEQ ID NO: 8)) and a PE comprising SEQ ID NO: 286(T15-LO10R+456A). In a preferred embodiment, the chimeric moleculecomprises the antigen binding portion of an anti-CAIX antibody, a linkercomprising the amino acid sequence of SEQ ID NO: 36, and a PE comprisingSEQ ID NO: 286 (T15-L010R+456A). In a preferred embodiment, the antigenbinding portion is the Fab fragment of an anti-CAIX antibody comprisingthe CDR1, CDR2 and CDR3 of the variable heavy chain domain VH of SEQ IDNO: 125 (SEQ ID NOs: 129, 133, and 137, respectively) and the CDR1, CDR2and CDR3 of the variable light chain domain of SEQ ID NO: 126 (SEQ IDNOs: 130, 134, and 138, respectively).

In a preferred embodiment, the chimeric molecule comprises the antigenbinding portion of an anti-CAIX antibody, a furin cleavage sequence(FCS) corresponding to amino acid residues 274-284 of SEQ ID NO: 1(RHRQPRGWEQL (SEQ ID NO: 8)) and a PE comprising SEQ ID NO: 287(T14-LO10R). In a preferred embodiment, the chimeric molecule comprisesthe antigen binding portion of an anti-CAIX antibody, a linkercomprising the amino acid sequence of SEQ ID NO: 36, and a PE comprisingSEQ ID NO: 287 (T14-L010R). In a preferred embodiment, the antigenbinding portion is the Fab fragment of an anti-CAIX antibody comprisingthe CDR1, CDR2 and CDR3 of the variable heavy chain domain VH of SEQ IDNO: 125 (SEQ ID NOs: 129, 133, and 137, respectively) and the CDR1, CDR2and CDR3 of the variable light chain domain of SEQ ID NO: 126 (SEQ IDNOs: 130, 134, and 138, respectively).

In a preferred embodiment, the chimeric molecule comprises the antigenbinding portion of an anti-CAIX antibody, a furin cleavage sequence(FCS) corresponding to amino acid residues 274-284 of SEQ ID NO: 1(RHRQPRGWEQL (SEQ ID NO: 8)) and a PE comprising SEQ ID NO: 288(T15-LO10R). In a preferred embodiment, the chimeric molecule comprisesthe antigen binding portion of an anti-CAIX antibody, a linkercomprising the amino acid sequence of SEQ ID NO: 36, and a PE comprisingSEQ ID NO: 288 (T15-L010R). In a preferred embodiment, the antigenbinding portion is the Fab fragment of an anti-CAIX antibody comprisingthe CDR1, CDR2 and CDR3 of the variable heavy chain domain VH of SEQ IDNO: 125 (SEQ ID NOs: 129, 133, and 137, respectively) and the CDR1, CDR2and CDR3 of the variable light chain domain of SEQ ID NO: 126 (SEQ IDNOs: 130, 134, and 138, respectively). In a preferred embodiment, thechimeric molecule comprises the antigen binding portion of an anti-L1CAMantibody, a furin cleavage sequence (FCS) corresponding to amino acidresidues 274-284 of SEQ ID NO: 1 (RHRQPRGWEQL (SEQ ID NO: 8)) and a PEcomprising SEQ ID NO: 289 (T18/T20). In a preferred embodiment, thechimeric molecule comprises the antigen binding portion of an anti-L1CAM antibody, a linker comprising the amino acid sequence of SEQ ID NO:36, and aPE comprising. In a preferred embodiment, the antigen bindingportion is the Fab fragment of an anti-L1CAM antibody comprising theCDR1, CDR2 and CDR3 of the variable heavy chain domain VH of SEQ ID NO:141 (SEQ ID NOs: 145, 149, and 153, respectively) and the CDR1, CDR2 andCDR3 of the variable light chain domain of SEQ ID NO: 142 (SEQ ID NOs:146, 150, and 154, respectively). In a preferred embodiment, the antigenbinding portion is the Fab fragment of an anti-L1 CAM antibodycomprising the variable heavy chain domain VH of SEQ ID NO: 157 and thevariable light chain domain of SEQ ID NO: 158. In a preferredembodiment, the antigen binding portion is the Fab fragment of ananti-L1 CAM antibody comprising the variable heavy chain domain VH ofSEQ ID NO: 173 and the variable light chain domain of SEQ ID NO: 174.

In a preferred embodiment, the chimeric molecule comprises the antigenbinding portion of an anti-L1CAM antibody, a furin cleavage sequence(FCS) corresponding to amino acid residues 274-284 of SEQ ID NO: 1(RHRQPRGWEQL (SEQ ID NO: 8)) and a PE comprising SEQ ID NO: 285(T14-LO10R+456A). In a preferred embodiment, the chimeric moleculecomprises the antigen binding portion of an anti-L1CAM antibody, alinker comprising the amino acid sequence of SEQ ID NO: 36, and a PEcomprising SEQ ID NO: 285 (T14-L010R+456A). In a preferred embodiment,the antigen binding portion is the Fab fragment of an anti-L1CAMantibody comprising the CDR1, CDR2 and CDR3 of the variable heavy chaindomain VH of SEQ ID NO: 141 (SEQ ID NOs: 145, 149, and 153,respectively) and the CDR1, CDR2 and CDR3 of the variable light chaindomain of SEQ ID NO: 142 (SEQ ID NOs: 146, 150, and 154, respectively).In a preferred embodiment, the antigen binding portion is the Fabfragment of an anti-L1CAM antibody comprising the variable heavy chaindomain VH of SEQ ID NO: 157 and the variable light chain domain of SEQID NO: 158. In a preferred embodiment, the antigen binding portion isthe Fab fragment of an anti-L1CAM antibody comprising the variable heavychain domain VH of SEQ ID NO: 173 and the variable light chain domain ofSEQ ID NO: 174.

In a preferred embodiment, the chimeric molecule comprises the antigenbinding portion of an anti-L1CAM antibody, a furin cleavage sequence(FCS) corresponding to amino acid residues 274-284 of SEQ ID NO: 1(RHRQPRGWEQL (SEQ ID NO: 8)) and a PE comprising SEQ ID NO: 286(T15-LO10R+456A). In a preferred embodiment, the chimeric moleculecomprises the antigen binding portion of an anti-L1CAM antibody, alinker comprising the amino acid sequence of SEQ ID NO: 36, and a PEcomprising SEQ ID NO: 286 (T15-L010R+456A). In a preferred embodiment,the antigen binding portion is the Fab fragment of an anti-L1CAMantibody comprising the CDR1, CDR2 and CDR3 of the variable heavy chaindomain VH of SEQ ID NO: 141 (SEQ ID NOs: 145, 149, and 153,respectively) and the CDR1, CDR2 and CDR3 of the variable light chaindomain of SEQ ID NO: 142 (SEQ ID NOs: 146, 150, and 154, respectively).In a preferred embodiment, the antigen binding portion is the Fabfragment of an anti-L1CAM antibody comprising the variable heavy chaindomain VH of SEQ ID NO: 157 and the variable light chain domain of SEQID NO: 158. In a preferred embodiment, the antigen binding portion isthe Fab fragment of an anti-L1CAM antibody comprising the variable heavychain domain VH of SEQ ID NO: 173 and the variable light chain domain ofSEQ ID NO: 174.

In a preferred embodiment, the chimeric molecule comprises the antigenbinding portion of an anti-L1CAM antibody, a furin cleavage sequence(FCS) corresponding to amino acid residues 274-284 of SEQ ID NO: 1(RHRQPRGWEQL (SEQ ID NO: 8)) and a PE comprising SEQ ID NO: 287(T14-LO10R). In a preferred embodiment, the chimeric molecule comprisesthe antigen binding portion of an anti-L1CAM antibody, a linkercomprising the amino acid sequence of SEQ ID NO: 36, and a PE comprisingSEQ ID NO: 287 (T14-L010R). In a preferred embodiment, the antigenbinding portion is the Fab fragment of an anti-L1CAM antibody comprisingthe CDR1, CDR2 and CDR3 of the variable heavy chain domain VH of SEQ IDNO: 141 (SEQ ID NOs: 145, 149, and 153, respectively) and the CDR1, CDR2and CDR3 of the variable light chain domain of SEQ ID NO: 142 (SEQ IDNOs: 146, 150, and 154, respectively). In a preferred embodiment, theantigen binding portion is the Fab fragment of an anti-L1CAM antibodycomprising the variable heavy chain domain VH of SEQ ID NO: 157 and thevariable light chain domain of SEQ ID NO: 158. In a preferredembodiment, the antigen binding portion is the Fab fragment of ananti-L1CAM antibody comprising the variable heavy chain domain VH of SEQID NO: 173 and the variable light chain domain of SEQ ID NO: 174.

In a preferred embodiment, the chimeric molecule comprises the antigenbinding portion of an anti-L1CAM antibody, a furin cleavage sequence(FCS) corresponding to amino acid residues 274-284 of SEQ ID NO: 1(RHRQPRGWEQL (SEQ ID NO: 8)) and a PE comprising SEQ ID NO: 288(T15-LO10R). In a preferred embodiment, the chimeric molecule comprisesthe antigen binding portion of an anti-L1CAM antibody, a linkercomprising the amino acid sequence of SEQ ID NO: 36, and a PE comprisingSEQ ID NO: 288 (T15-L010R). In a preferred embodiment, the antigenbinding portion is the Fab fragment of an anti-L1CAM antibody comprisingthe CDR1, CDR2 and CDR3 of the variable heavy chain domain VH of SEQ IDNO: 141 (SEQ ID NOs: 145, 149, and 153, respectively) and the CDR1, CDR2and CDR3 of the variable light chain domain of SEQ ID NO: 142 (SEQ IDNOs: 146, 150, and 154, respectively). In a preferred embodiment, theantigen binding portion is the Fab fragment of an anti-L1CAM antibodycomprising the variable heavy chain domain VH of SEQ ID NO: 157 and thevariable light chain domain of SEQ ID NO: 158. In a preferredembodiment, the antigen binding portion is the Fab fragment of ananti-L1CAM antibody comprising the variable heavy chain domain VH of SEQID NO: 173 and the variable light chain domain of SEQ ID NO: 174.Included in the scope of the invention are functional portions of theinventive PEs and chimeric molecules described herein. The term“functional portion” when used in reference to a PE or chimeric moleculerefers to any part or fragment of the PE or chimeric molecule of theinvention, which part or fragment retains the biological activity of thePE or chimeric molecule of which it is a part (the parent PE or chimericmolecule). Functional portions encompass, for example, those parts of aPE or chimeric molecule that retain the ability to specifically bind toand destroy or inhibit the growth of target cells or treat or preventcancer, to a similar extent, the same extent, or to a higher extent, asthe parent PE or chimeric molecule. In reference to the parent PE orchimeric molecule, the functional portion can comprise, for instance,about 10% or more, about 25% or more, about 30% or more, about 50% ormore, about 68% or more, about 80% or more, about 90% or more, or about95% or more, of the parent PE or chimeric molecule.

The functional portion can comprise additional amino acids at the aminoor carboxy terminus of the portion, or at both termini, which additionalamino acids are not found in the amino acid sequence of the parent PE orchimeric molecule. Desirably, the additional amino acids do notinterfere with the biological function of the functional portion, e.g.,specifically binding to and destroying or inhibiting the growth oftarget cells, having the ability to treat or prevent cancer, etc. Moredesirably, the additional amino acids enhance the biological activity,as compared to the biological activity of the parent PE or chimericmolecule.

Included in the scope of the invention are functional variants of theinventive PEs and chimeric molecules described herein. The term“functional variant” as used herein refers to a PE or chimeric moleculehaving substantial or significant sequence identity or similarity to aparent PE or chimeric molecule, which functional variant retains thebiological activity of the PE or chimeric molecule of which it is avariant. Functional variants encompass, for example, those variants ofthe PE or chimeric molecule described herein (the parent PE or chimericmolecule) that retain the ability to specifically bind to and destroy orinhibit the growth of target cells to a similar extent, the same extent,or to a higher extent, as the parent PE or chimeric molecule. Inreference to the parent PE or chimeric molecule, the functional variantcan, for instance, be about 30% or more, about 50% or more, about 75% ormore, about 80% or more, about 90% or more, about 95% or more, about 96%or more, about 97% or more, about 98% or more, or about 99% or moreidentical in amino acid sequence to the parent PE or chimeric molecule.

The functional variant can, for example, comprise the amino acidsequence of the parent PE or chimeric molecule with at least oneconservative amino acid substitution. Conservative amino acidsubstitutions are known in the art and include amino acid substitutionsin which one amino acid having certain chemical and/or physicalproperties is exchanged for another amino acid that has the samechemical or physical properties. For instance, the conservative aminoacid substitution can be an acidic amino acid substituted for anotheracidic amino acid (e.g., Asp or Glu), an amino acid with a nonpolar sidechain substituted for another amino acid with a nonpolar side chain(e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), a basicamino acid substituted for another basic amino acid (Lys, Arg, etc.), anamino acid with a polar side chain substituted for another amino acidwith a polar side chain (Asn, Cys, Gln, Ser, Thr, Tyr, etc.), etc.

Alternatively or additionally, the functional variants can comprise theamino acid sequence of the parent PE or chimeric molecule with at leastone non-conservative amino acid substitution. In this case, it ispreferable for the non-conservative amino acid substitution to notinterfere with or inhibit the biological activity of the functionalvariant. Preferably, the non-conservative amino acid substitutionenhances the biological activity of the functional variant, such thatthe biological activity of the functional variant is increased ascompared to the parent PE or chimeric molecule.

The PE or chimeric molecule of the invention can consist essentially ofthe specified amino acid sequence or sequences described herein, suchthat other components of the functional variant, e.g., other aminoacids, do not materially change the biological activity of thefunctional variant.

The PE or chimeric molecule of the invention (including functionalportions and functional variants) of the invention can comprisesynthetic amino acids in place of one or more naturally-occurring aminoacids. Such synthetic amino acids are known in the art and include, forexample, aminocyclohexane carboxylic acid, norleucine, α-aminon-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- andtrans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine,4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserineβ-hydroxyphenylalanine, phenylglycine, α-naphthylalanine,cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid,1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid,aminomalonic acid monoamide, N′-benzyl-N′-methyl-lysine,N′,N′-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexane carboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid,α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine,and α-tert-butylglycine.

The PE or chimeric molecule of the invention (including functionalportions and functional variants) can be glycosylated, amidated,carboxylated, phosphorylated, esterified, N-acylated, cyclized via,e.g., a disulfide bridge, or converted into an acid addition salt and/oroptionally dimerized or polymerized, or conjugated.

An embodiment of the invention provides a method of producing theinventive PE comprising (a) recombinantly expressing the PE and (b)purifying the PE. The PEs and chimeric molecules of the invention(including functional portions and functional variants) can be obtainedby methods of producing proteins and polypeptides known in the art.Suitable methods of de novo synthesizing polypeptides and proteins aredescribed in references, such as Chan et al., Fmoc Solid Phase PeptideSynthesis, Oxford University Press, Oxford, United Kingdom, 2005;Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker, Inc.,2000; Epitope Mapping, ed. Westwood et al., Oxford University Press,Oxford, United Kingdom, 2000; and U.S. Pat. No. 5,449,752. Also, the PEsand chimeric molecules of the invention can be recombinantly expressedusing the nucleic acids described herein using standard recombinantmethods. See, for instance, Sambrook et al., Molecular Cloning: ALaboratory Manual, 3^(rd) ed., Cold Spring Harbor Press, Cold SpringHarbor, N.Y. 2001; and Ausubel et al., Current Protocols in MolecularBiology, Greene Publishing Associates and John Wiley & Sons, N Y, 1994.

The method further comprises purifying the PE. Once expressed, theinventive PEs may be purified in accordance with purification techniquesknown in the art. Exemplary purification techniques include, but are notlimited to, ammonium sulfate precipitation, affinity columns, and columnchromatography, or by procedures described in, e.g., R. Scopes, ProteinPurification, Springer-Verlag, N.Y. (1982).

Another embodiment of the invention provides a method of producing theinventive chimeric molecule comprising (a) recombinantly expressing thechimeric molecule and (b) purifying the chimeric molecule. The chimericmolecule may be recombinantly expressed and purified as described hereinwith respect to other aspects of the invention. In an embodiment of theinvention, recombinantly expressing the chimeric molecule comprisesinserting a nucleotide sequence encoding a targeting moiety and anucleotide sequence encoding a PE into a vector. The method may compriseinserting the nucleotide sequence encoding the targeting moiety and thenucleotide sequence encoding the PE in frame so that it encodes onecontinuous polypeptide including a functional targeting moiety regionand a functional PE region. In an embodiment of the invention, themethod comprises ligating a nucleotide sequence encoding the PE to anucleotide sequence encoding a targeting moiety so that, uponexpression, the PE is located at the carboxyl terminus of the targetingmoiety. In an alternative embodiment, the method comprises ligating anucleotide sequence encoding the PE to a nucleotide sequence encoding atargeting moiety so that, upon expression, the PE is located at theamino terminus of the targeting moiety.

Still another embodiment of the invention provides a method of producingthe inventive chimeric molecule comprising (a) recombinantly expressingthe inventive PE, (b) purifying the PE, and (c) covalently linking atargeting moiety to the purified PE. The inventive PE may berecombinantly expressed as described herein with respect to otheraspects of the invention. The method further comprises covalentlylinking a targeting moiety to the purified PE. The method of attaching aPE to a targeting moiety may vary according to the chemical structure ofthe targeting moiety. For example, the method may comprise reacting anyone or more of a variety of functional groups e.g., carboxylic acid(COOH), free amine (—NH₂), or sulfhydryl (—SH) groups present on the PEwith a suitable functional group on the targeting moiety, therebyforming a covalent bind between the PE and the targeting moiety.Alternatively or additionally, the method may comprise derivatizing thetargeting moiety or PE to expose or to attach additional reactivefunctional groups. Derivatizing may also include attaching one or morelinkers to the targeting moiety or PE.

In another embodiment of the invention, the inventive PEs and chimericmolecules may be produced using non-recombinant methods. For example,the inventive PEs and chimeric molecules described herein (includingfunctional portions and functional variants) can be commerciallysynthesized by companies, such as Synpep (Dublin, Calif.), PeptideTechnologies Corp. (Gaithersburg, Md.), and Multiple Peptide Systems(San Diego, Calif.). In this respect, the inventive PEs and chimericmolecules can be synthetic, recombinant, isolated, and/or purified.

It may be desirable, in some circumstances, to free the PE from thetargeting moiety when the chimeric molecule has reached one or moretarget cells. In this regard, the inventive chimeric molecules maycomprise a cleavable linker. The linker may be cleavable by any suitablemeans, e.g., enzymatically. For example, when the target cell is acancer (e.g., tumor) cell, the chimeric molecule may include a linkercleavable under conditions present at the tumor site (e.g., when exposedto tumor-associated enzymes or acidic pH).

An embodiment of the invention provides a nucleic acid comprising anucleotide sequence encoding any of the inventive PEs or the inventivechimeric molecules described herein. The term “nucleic acid,” as usedherein, includes “polynucleotide,” “oligonucleotide,” and “nucleic acidmolecule,” and generally means a polymer of DNA or RNA, which can besingle-stranded or double-stranded, which can be synthesized or obtained(e.g., isolated and/or purified) from natural sources, which can containnatural, non-natural or altered nucleotides, and which can contain anatural, non-natural, or altered internucleotide linkage, such as aphosphoroamidate linkage or a phosphorothioate linkage, instead of thephosphodiester found between the nucleotides of an unmodifiedoligonucleotide. It is generally preferred that the nucleic acid doesnot comprise any insertions, deletions, inversions, and/orsubstitutions. However, it may be suitable in some instances, asdiscussed herein, for the nucleic acid to comprise one or moreinsertions, deletions, inversions, and/or substitutions.

Preferably, the nucleic acids of the invention are recombinant. As usedherein, the term “recombinant” refers to (i) molecules that areconstructed outside living cells by joining natural or synthetic nucleicacid segments, or (ii) molecules that result from the replication ofthose described in (i) above. For purposes herein, the replication canbe in vitro replication or in vivo replication.

The nucleic acids can be constructed based on chemical synthesis and/orenzymatic ligation reactions using procedures known in the art. See, forexample, Sambrook et al., supra, and Ausubel et al., supra. For example,a nucleic acid can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed upon hybridization (e.g.,phosphorothioate derivatives and acridine substituted nucleotides).Examples of modified nucleotides that can be used to generate thenucleic acids include, but are not limited to, 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxymethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N⁶-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N⁶-substitutedadenine, 7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N⁶-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleicacids of the invention can be purchased from companies, such asMacromolecular Resources (Fort Collins, Colo.) and Synthegen (Houston,Tex.).

The invention also provides a nucleic acid comprising a nucleotidesequence which is complementary to the nucleotide sequence of any of thenucleic acids described herein or a nucleotide sequence which hybridizesunder stringent conditions to the nucleotide sequence of any of thenucleic acids described herein.

The nucleotide sequence which hybridizes under stringent conditionspreferably hybridizes under high stringency conditions. By “highstringency conditions” is meant that the nucleotide sequencespecifically hybridizes to a target sequence (the nucleotide sequence ofany of the nucleic acids described herein) in an amount that isdetectably stronger than non-specific hybridization. High stringencyconditions include conditions which would distinguish a polynucleotidewith an exact complementary sequence, or one containing only a fewscattered mismatches, from a random sequence that happened to have onlya few small regions (e.g., 3-10 bases) that matched the nucleotidesequence. Such small regions of complementarity are more easily meltedthan a full-length complement of 14-17 or more bases, and highstringency hybridization makes them easily distinguishable. Relativelyhigh stringency conditions would include, for example, low salt and/orhigh temperature conditions, such as provided by about 0.02-0.1 M NaClor the equivalent, at temperatures of about 50-70° C. Such highstringency conditions tolerate little, if any, mismatch between thenucleotide sequence and the template or target strand, and areparticularly suitable for detecting expression of any of the inventivePEs or chimeric molecules. It is generally appreciated that conditionscan be rendered more stringent by the addition of increasing amounts offormamide.

The invention also provides a nucleic acid comprising a nucleotidesequence that is about 70% or more, e.g., about 80% or more, about 90%or more, about 91% or more, about 92% or more, about 93% or more, about94% or more, about 95% or more, about 96% or more, about 97% or more,about 98% or more, or about 99% or more identical to any of the nucleicacids described herein.

The nucleic acids of the invention can be incorporated into arecombinant expression vector. In this regard, the invention providesrecombinant expression vectors comprising any of the nucleic acids ofthe invention. For purposes herein, the term “recombinant expressionvector” means a genetically-modified oligonucleotide or polynucleotideconstruct that permits the expression of an mRNA, protein, polypeptide,or peptide by a host cell, when the construct comprises a nucleotidesequence encoding the mRNA, protein, polypeptide, or peptide, and thevector is contacted with the cell under conditions sufficient to havethe mRNA, protein, polypeptide, or peptide expressed within the cell.The vectors of the invention are not naturally-occurring as a whole.However, parts of the vectors can be naturally-occurring. The inventiverecombinant expression vectors can comprise any type of nucleotides,including, but not limited to DNA and RNA, which can be single-strandedor double-stranded, which can be synthesized or obtained in part fromnatural sources, and which can contain natural, non-natural or alterednucleotides. The recombinant expression vectors can comprisenaturally-occurring, non-naturally-occurring internucleotide linkages,or both types of linkages. Preferably, the non-naturally occurring oraltered nucleotides or internucleotide linkages does not hinder thetranscription or replication of the vector.

The recombinant expression vector of the invention can be any suitablerecombinant expression vector, and can be used to transform or transfectany suitable host cell. Suitable vectors include those designed forpropagation and expansion or for expression or for both, such asplasmids and viruses. The vector can be selected from the groupconsisting of the pUC series (Fermentas Life Sciences), the pBluescriptseries (Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison,Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEXseries (Clontech, Palo Alto, Calif.). Bacteriophage vectors, such asλGT10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149, also can beused. Examples of plant expression vectors include pBI01, pBI101.2,pBI101.3, pBI121 and pBIN19 (Clontech). Examples of animal expressionvectors include pEUK-C1, pMAM, and pMAMneo (Clontech). Preferably, therecombinant expression vector is a viral vector, e.g., a retroviralvector.

The recombinant expression vectors of the invention can be preparedusing standard recombinant DNA techniques described in, for example,Sambrook et al., supra, and Ausubel et al., supra. Constructs ofexpression vectors, which are circular or linear, can be prepared tocontain a replication system functional in a prokaryotic or eukaryotichost cell. Replication systems can be derived, e.g., from ColE1, 2μplasmid, λ, SV40, bovine papilloma virus, and the like.

Desirably, the recombinant expression vector comprises regulatorysequences, such as transcription and translation initiation andtermination codons, which are specific to the type of host (e.g.,bacterium, fungus, plant, or animal) into which the vector is to beintroduced, as appropriate and taking into consideration whether thevector is DNA- or RNA-based.

The recombinant expression vector can include one or more marker genes,which allow for selection of transformed or transfected hosts. Markergenes include biocide resistance, e.g., resistance to antibiotics, heavymetals, etc., complementation in an auxotrophic host to provideprototrophy, and the like. Suitable marker genes for the inventiveexpression vectors include, for instance, neomycin/G418 resistancegenes, hygromycin resistance genes, histidinol resistance genes,tetracycline resistance genes, and ampicillin resistance genes.

The recombinant expression vector can comprise a native or nonnativepromoter operably linked to the nucleotide sequence encoding theinventive PE or chimeric molecule (including functional portions andfunctional variants), or to the nucleotide sequence which iscomplementary to or which hybridizes to the nucleotide sequence encodingthe PE or chimeric molecule. The selection of promoters, e.g., strong,weak, inducible, tissue-specific, and developmental-specific, is withinthe ordinary skill of the artisan. Similarly, the combining of anucleotide sequence with a promoter is also within the ordinary skill ofthe artisan. The promoter can be a non-viral promoter or a viralpromoter, e.g., a cytomegalovirus (CMV) promoter, an SV40 promoter, anRSV promoter, or a promoter found in the long-terminal repeat of themurine stem cell virus.

The inventive recombinant expression vectors can be designed for eithertransient expression, for stable expression, or for both. Also, therecombinant expression vectors can be made for constitutive expressionor for inducible expression.

Another embodiment of the invention further provides a host cellcomprising any of the recombinant expression vectors described herein.As used herein, the term “host cell” refers to any type of cell that cancontain the inventive recombinant expression vector. The host cell canbe a eukaryotic cell, e.g., plant, animal, fungi, or algae, or can be aprokaryotic cell, e.g., bacteria or protozoa. The host cell can be acultured cell, an adherent cell or a suspended cell, i.e., a cell thatgrows in suspension. For purposes of producing a recombinant inventivePE or chimeric molecule, the host cell is preferably a prokaryotic cell,e.g., an E. coli cell.

Also provided by the invention is a population of cells comprising atleast one host cell described herein. The population of cells can be aheterogeneous population comprising the host cell comprising any of therecombinant expression vectors described, in addition to at least oneother cell, e.g., a host cell which does not comprise any of therecombinant expression vectors. Alternatively, the population of cellscan be a substantially homogeneous population, in which the populationcomprises mainly (e.g., consisting essentially of) host cells comprisingthe recombinant expression vector. The population also can be a clonalpopulation of cells, in which all cells of the population are clones ofa single host cell comprising a recombinant expression vector, such thatall cells of the population comprise the recombinant expression vector.In one embodiment of the invention, the population of cells is a clonalpopulation of host cells comprising a recombinant expression vector asdescribed herein.

The inventive PEs, chimeric molecules (including functional portions andfunctional variants), nucleic acids, recombinant expression vectors,host cells (including populations thereof), and populations of cells canbe isolated and/or purified. The term “isolated” as used herein meanshaving been removed from its natural environment. The term “purified” asused herein means having been increased in purity, wherein “purity” is arelative term, and not to be necessarily construed as absolute purity.For example, the purity can be about 50% or more, about 60% or more,about 70% or more, about 80% or more, about 90% or more, or about 100%.The purity preferably is about 90% or more (e.g., about 90% to about95%) and more preferably about 98% or more (e.g., about 98% to about99%).

The inventive PEs, chimeric molecules (including functional portions andfunctional variants), nucleic acids, recombinant expression vectors,host cells (including populations thereof), and populations of cells,all of which are collectively referred to as “inventive PE materials”hereinafter, can be formulated into a composition, such as apharmaceutical composition. In this regard, the invention provides apharmaceutical composition comprising any of the PEs, chimeric molecules(including functional portions and functional variants), nucleic acids,recombinant expression vectors, host cells (including populationsthereof), and populations of cells, and a pharmaceutically acceptablecarrier. The inventive pharmaceutical composition containing any of theinventive PE materials can comprise more than one inventive PE material,e.g., a polypeptide and a nucleic acid, or two or more different PEs.Alternatively, the pharmaceutical composition can comprise an inventivePE material in combination with one or more other pharmaceuticallyactive agents or drugs, such as a chemotherapeutic agents, e.g.,asparaginase, busulfan, carboplatin, cisplatin, daunorubicin,doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate,paclitaxel, rituximab, vinblastine, vincristine, etc.

Preferably, the carrier is a pharmaceutically acceptable carrier. Withrespect to pharmaceutical compositions, the carrier can be any of thoseconventionally used and is limited only by chemico-physicalconsiderations, such as solubility and lack of reactivity with theactive compound(s), and by the route of administration. Thepharmaceutically acceptable carriers described herein, for example,vehicles, adjuvants, excipients, and diluents, are well-known to thoseskilled in the art and are readily available to the public. It ispreferred that the pharmaceutically acceptable carrier be one which ischemically inert to the active agent(s) and one which has no detrimentalside effects or toxicity under the conditions of use.

The choice of carrier will be determined in part by the particularinventive PE material, as well as by the particular method used toadminister the inventive PE material. Accordingly, there are a varietyof suitable formulations of the pharmaceutical composition of theinvention. The following formulations for parenteral (e.g.,subcutaneous, intravenous, intraarterial, intramuscular, intradermal,interperitoneal, and intrathecal), oral, and aerosol administration areexemplary and are in no way limiting. More than one route can be used toadminister the inventive PE materials, and in certain instances, aparticular route can provide a more immediate and more effectiveresponse than another route.

Topical formulations are well-known to those of skill in the art. Suchformulations are particularly suitable in the context of the inventionfor application to the skin.

Formulations suitable for oral administration can include (a) liquidsolutions, such as an effective amount of the inventive PE materialdissolved in diluents, such as water, saline, or orange juice; (b)capsules, sachets, tablets, lozenges, and troches, each containing apredetermined amount of the active ingredient, as solids or granules;(c) powders; (d) suspensions in an appropriate liquid; and (e) suitableemulsions. Liquid formulations may include diluents, such as water andalcohols, for example, ethanol, benzyl alcohol, and the polyethylenealcohols, either with or without the addition of a pharmaceuticallyacceptable surfactant. Capsule forms can be of the ordinary hard- orsoft-shelled gelatin type containing, for example, surfactants,lubricants, and inert fillers, such as lactose, sucrose, calciumphosphate, and corn starch. Tablet forms can include one or more oflactose, sucrose, mannitol, corn starch, potato starch, alginic acid,microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicondioxide, croscarmellose sodium, talc, magnesium stearate, calciumstearate, zinc stearate, stearic acid, and other excipients, colorants,diluents, buffering agents, disintegrating agents, moistening agents,preservatives, flavoring agents, and other pharmacologically compatibleexcipients. Lozenge forms can comprise the inventive PE material in aflavor, usually sucrose and acacia or tragacanth, as well as pastillescomprising the inventive PE material in an inert base, such as gelatinand glycerin, or sucrose and acacia, emulsions, gels, and the likeadditionally containing such excipients as are known in the art.

The inventive PE material, alone or in combination with other suitablecomponents, can be made into aerosol formulations to be administered viainhalation. These aerosol formulations can be placed into pressurizedacceptable propellants, such as dichlorodifluoromethane, propane,nitrogen, and the like. The aerosol formulations also may be formulatedas pharmaceuticals for non-pressured preparations, such as in anebulizer or an atomizer. Such spray formulations also may be used tospray mucosa.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The inventive PE material can be administered in a physiologicallyacceptable diluent in a pharmaceutical carrier, such as a sterile liquidor mixture of liquids, including water, saline, aqueous dextrose andrelated sugar solutions, an alcohol, such as ethanol or hexadecylalcohol, a glycol, such as propylene glycol or polyethylene glycol,dimethylsulfoxide, glycerol, ketals such as2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, poly(ethyleneglycol) 400,oils, fatty acids, fatty acid esters or glycerides, or acetylated fattyacid glycerides with or without the addition of a pharmaceuticallyacceptable surfactant, such as a soap or a detergent, suspending agent,such as pectin, carbomers, methylcellulose,hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifyingagents and other pharmaceutical adjuvants.

Oils, which can be used in parenteral formulations include petroleum,animal, vegetable, or synthetic oils. Specific examples of oils includepeanut, soybean, sesame, cottonseed, corn, olive, petrolatum, andmineral. Suitable fatty acids for use in parenteral formulations includeoleic acid, stearic acid, and isostearic acid. Ethyl oleate andisopropyl myristate are examples of suitable fatty acid esters.

Suitable soaps for use in parenteral formulations include fatty alkalimetal, ammonium, and triethanolamine salts, and suitable detergentsinclude (a) cationic detergents such as, for example, dimethyl dialkylammonium halides, and alkyl pyridinium halides, (b) anionic detergentssuch as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin,ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionicdetergents such as, for example, fatty amine oxides, fatty acidalkanolamides, and polyoxyethylenepolypropylene copolymers, (d)amphoteric detergents such as, for example, alkyl-β-aminopropionates,and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixturesthereof.

The parenteral formulations will typically contain from about 0.5% toabout 25% by weight of the inventive PE material in solution.Preservatives and buffers may be used. In order to minimize or eliminateirritation at the site of injection, such compositions may contain oneor more nonionic surfactants having a hydrophile-lipophile balance (HLB)of from about 12 to about 17. The quantity of surfactant in suchformulations will typically range from about 5% to about 15% by weight.Suitable surfactants include polyethylene glycol sorbitan fatty acidesters, such as sorbitan monooleate and the high molecular weightadducts of ethylene oxide with a hydrophobic base, formed by thecondensation of propylene oxide with propylene glycol. The parenteralformulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampoules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tabletsof the kind previously described. The requirements for effectivepharmaceutical carriers for parenteral compositions are well-known tothose of ordinary skill in the art (see, e.g., Pharmaceutics andPharmacy Practice, J. B. Lippincott Company, Philadelphia, Pa., Bankerand Chalmers, eds., pages 238-250 (1982), and ASHP Handbook onInjectable Drugs, Toissel, 4th ed., pages 622-630 (1986)).

It will be appreciated by one of skill in the art that, in addition tothe above-described pharmaceutical compositions, the inventive PEmaterials of the invention can be formulated as inclusion complexes,such as cyclodextrin inclusion complexes, or liposomes.

For purposes of the invention, the amount or dose of the inventive PEmaterial administered should be sufficient to effect a desired response,e.g., a therapeutic or prophylactic response, in the mammal over areasonable time frame. For example, the dose of the inventive PEmaterial should be sufficient to inhibit growth of a target cell ortreat or prevent cancer in a period of from about 2 hours or longer,e.g., 12 to 24 or more hours, from the time of administration. Incertain embodiments, the time period could be even longer. The dose willbe determined by the efficacy of the particular inventive PE materialand the condition of the mammal (e.g., human), as well as the bodyweight of the mammal (e.g., human) to be treated.

Many assays for determining an administered dose are known in the art.An administered dose may be determined in vitro (e.g., cell cultures) orin vivo (e.g., animal studies). For example, an administered dose may bedetermined by determining the IC₅₀ (the dose that achieves ahalf-maximal inhibition of symptoms), LD₅₀ (the dose lethal to 50% ofthe population), the ED₅₀ (the dose therapeutically effective in 50% ofthe population), and the therapeutic index in cell culture and/or animalstudies. The therapeutic index is the ratio of LD₅₀ to ED₅₀ (i.e.,LD₅₀/ED₅₀).

The dose of the inventive PE material also will be determined by theexistence, nature, and extent of any adverse side effects that mightaccompany the administration of a particular inventive PE material.Typically, the attending physician will decide the dosage of theinventive PE material with which to treat each individual patient,taking into consideration a variety of factors, such as age, bodyweight, general health, diet, sex, inventive PE material to beadministered, route of administration, and the severity of the conditionbeing treated. By way of example and not intending to limit theinvention, the dose of the inventive PE material can be about 0.001 toabout 1000 mg/kg body weight of the subject being treated/day, fromabout 0.01 to about 10 mg/kg body weight/day, about 0.01 mg to about 1mg/kg body weight/day, from about 1 to about to about 1000 mg/kg bodyweight/day, from about 5 to about 500 mg/kg body weight/day, from about10 to about 250 mg/kg body weight/day, about 25 to about 150 mg/kg bodyweight/day, or about 10 mg/kg body weight/day.

Alternatively, the inventive PE materials can be modified into a depotform, such that the manner in which the inventive PE material isreleased into the body to which it is administered is controlled withrespect to time and location within the body (see, for example, U.S.Pat. No. 4,450,150). Depot forms of inventive PE materials can be, forexample, an implantable composition comprising the inventive PEmaterials and a porous or non-porous material, such as a polymer,wherein the inventive PE materials is encapsulated by or diffusedthroughout the material and/or degradation of the non-porous material.The depot is then implanted into the desired location within the bodyand the inventive PE materials are released from the implant at apredetermined rate.

The inventive PE materials may be assayed for cytoxicity by assays knownin the art. Examples of cytotoxicity assays include a WST assay, whichmeasures cell proliferation using the tetrazolium salt WST-1 (reagentsand kits available from Roche Applied Sciences), as described inInternational Patent Application Publication WO 2011/032022.

It is contemplated that the inventive pharmaceutical compositions, PEs,chimeric molecules, nucleic acids, recombinant expression vectors, hostcells, or populations of cells can be used in methods of treating orpreventing cancer. Without being bound by a particular theory ormechanism, it is believed that the inventive PEs destroy or inhibit thegrowth of cells through the inhibition of protein synthesis ineukaryotic cells, e.g., by the inactivation of the ADP-ribosylation ofelongation factor 2 (EF-2). Without being bound to a particular theoryor mechanism, the inventive chimeric molecules recognize andspecifically bind to cell surface markers, thereby delivering thecytotoxic PE to the population of cells expressing the cell surfacemarker with minimal or no cross-reactivity with cells that do notexpress the cell surface marker. In this way, the cytoxicity of PE canbe targeted to destroy or inhibit the growth of a particular populationof cells, e.g., cancer cells. In this regard, the invention provides amethod of treating or preventing cancer in a mammal comprisingadministering to the mammal any of the PEs, chimeric molecules, nucleicacids, recombinant expression vectors, host cell, population of cells,or pharmaceutical compositions described herein, in an amount effectiveto treat or prevent cancer in the mammal.

The terms “treat” and “prevent” as well as words stemming therefrom, asused herein, do not necessarily imply 100% or complete treatment orprevention. Rather, there are varying degrees of treatment or preventionof which one of ordinary skill in the art recognizes as having apotential benefit or therapeutic effect. In this respect, the inventivemethods can provide any amount of any level of treatment or preventionof cancer in a mammal. Furthermore, the treatment or prevention providedby the inventive method can include treatment or prevention of one ormore conditions or symptoms of the disease, e.g., cancer, being treatedor prevented. Also, for purposes herein, “prevention” can encompassdelaying the onset of the disease, or a symptom or condition thereof

For purposes of the inventive methods, wherein host cells or populationsof cells are administered, the cells can be cells that are allogeneic orautologous to the host. Preferably, the cells are autologous to thehost.

With respect to the inventive methods, the cancer can be any cancer,including any of adrenal gland cancer, sarcomas (e.g., synovial sarcoma,osteogenic sarcoma, leiomyosarcoma uteri, angiosarcoma, fibrosarcoma,rhabdomyosarcoma, liposarcoma, myxoma, rhabdomyoma, fibroma, lipoma, andteratoma), lymphomas (e.g., small lymphocytic lymphoma, Hodgkinlymphoma, and non-Hodgkin lymphoma), hepatocellular carcinoma, glioma,head cancers (e.g., squamous cell carcinoma), neck cancers (e.g.,squamous cell carcinoma), acute lymphocytic cancer, leukemias (e.g.,hairy cell leukemia, myeloid leukemia (acute and chronic), lymphaticleukemia (acute and chronic), prolymphocytic leukemia (PLL),myelomonocytic leukemia (acute and chronic), and lymphocytic leukemia(acute and chronic)), bone cancer (osteogenic sarcoma, fibrosarcoma,malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma,malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignantgiant cell tumor, chordoma, osteochondroma (osteocartilaginousexostoses), benign chondroma, chondroblastoma, chondromyxoid fibroma,osteoid osteoma, and giant cell tumors), brain cancer (astrocytoma,medulloblastoma, glioma, ependymoma, germinoma (pinealoma), glioblastomamultiforme, oligodendroglioma, schwannoma, and retinoblastoma),fallopian tube cancer, breast cancer, cancer of the anus, anal canal, oranorectum, cancer of the eye, cancer of the intrahepatic bile duct,cancer of the joints, cancer of the neck, gallbladder, or pleura, cancerof the nose, nasal cavity, or middle ear, cancer of the oral cavity,cancer of the vulva (e.g., squamous cell carcinoma, intraepithelialcarcinoma, adenocarcinoma, and fibrosarcoma), myeloproliferativedisorders (e.g., chronic myeloid cancer), colon cancers (e.g., coloncarcinoma), esophageal cancer (e.g., squamous cell carcinoma,adenocarcinoma, leiomyosarcoma, and lymphoma), cervical cancer (cervicalcarcinoma and pre-invasive cervical dysplasia), gastric cancer,gastrointestinal carcinoid tumor, hypopharynx cancer, larynx cancer,liver cancers (e.g., hepatocellular carcinoma, cholangiocarcinoma,hepatoblastoma, angiosarcoma, hepatocellular adenoma, and hemangioma),lung cancers (e.g., bronchogenic carcinoma (squamous cell,undifferentiated small cell, undifferentiated large cell, andadenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma,chondromatous hamartoma, small cell lung cancer, non-small cell lungcancer, and lung adenocarcinoma), mesothelioma, skin cancer (e.g.,melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi'ssarcoma, nevi, dysplastic nevi, lipoma, angioma, dermatofibroma, andkeloids), multiple myeloma, nasopharynx cancer, ovarian cancer (e.g.,ovarian carcinoma (serous cystadenocarcinoma, mucinouscystadenocarcinoma, endometrioid carcinoma, and clear celladenocarcinoma), granulosa-theca cell tumors, Sertoli-Leydig celltumors, dysgerminoma, and malignant teratoma), pancreatic cancer (e.g.,ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoidtumors, and VIPoma), peritoneum, omentum, mesentery cancer, pharynxcancer, prostate cancer (e.g., adenocarcinoma and sarcoma), rectalcancer, kidney cancer (e.g., adenocarcinoma, Wilms tumor(nephroblastoma), and renal cell carcinoma), small intestine cancer(adenocarcinoma, lymphoma, carcinoid tumors, Kaposi's sarcoma,leiomyoma, hemangioma, lipoma, neurofibroma, and fibroma), soft tissuecancer, stomach cancer (e.g., carcinoma, lymphoma, and leiomyosarcoma),testicular cancer (e.g., seminoma, teratoma, embryonal carcinoma,teratocarcinoma, choriocarcinoma, sarcoma, Leydig cell tumor, fibroma,fibroadenoma, adenomatoid tumors, and lipoma), cancer of the uterus(e.g., endometrial carcinoma), thyroid cancer, and urothelial cancers(e.g., squamous cell carcinoma, transitional cell carcinoma,adenocarcinoma, ureter cancer, and urinary bladder cancer).

As used herein, the term “mammal” refers to any mammal, including, butnot limited to, mammals of the order Rodentia, such as mice andhamsters, and mammals of the order Logomorpha, such as rabbits. It ispreferred that the mammals are from the order Carnivora, includingFelines (cats) and Canines (dogs). It is more preferred that the mammalsare from the order Artiodactyla, including Bovines (cows) and Swines(pigs) or of the order Perssodactyla, including Equines (horses). It ismost preferred that the mammals are of the order Primates, Ceboids, orSimoids (monkeys) or of the order Anthropoids (humans and apes). Anespecially preferred mammal is the human.

Also provided is a method of inhibiting the growth of a target cellcomprising contacting the cell with the PE of any of the PEs, chimericmolecules, nucleic acids, recombinant expression vectors, host cell,population of cells, or pharmaceutical compositions described herein, inan amount effective to inhibit growth of the target cell. The growth ofthe target cell may be inhibited by any amount, e.g., by about 10% ormore, about 15% or more, about 20% or more, about 25% or more, about 30%or more, about 35% or more, about 40% or more, about 45% or more, about50% or more, about 55% or more, about 60% or more, about 65% or more,about 70% or more, about 75% or more, about 80% or more, about 85% ormore, about 90% or more, about 95% or more, or about 100%. The targetcell may be provided in a biological sample. A biological sample may beobtained from a mammal in any suitable manner and from any suitablesource. The biological sample may, for example, be obtained by a blooddraw, leukapheresis, and/or tumor biopsy or necropsy. The contacting cantake place in vitro or in vivo with respect to the mammal. Preferably,the contacting is in vitro.

In an embodiment of the invention, the target cell is a cancer cell. Thetarget cell may be a cancer cell of any of the cancers described herein.In an embodiment of the invention, the target may express a cell surfacemarker. The cell surface marker may be any cell surface marker describedherein with respect to other aspects of the invention. The cell surfacemarker may be, for example, selected from the group consisting of CD19,CD21, CD22, CD25, CD30, CD33, CD79b, transferrin receptor, EGF receptor,mesothelin, cadherin, and Lewis Y.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

Example 1

This example demonstrates the identification of T cell epitopes indomain III of PE38.

To identify the T cell epitopes in PE38, peripheral blood mononuclearcells (PBMCs) were first incubated with a recombinant immunotoxin (RIT)for 14 days to allow the RIT to be processed by donor APCs to relevantpeptides to be presented to T cells. The enriched PBMCs weresubsequently exposed to a peptide library composed of 111 partiallyoverlapping peptides spanning the sequence of PE38 and T cell activationwas measured using an ELISpot assay for interleukin-2 (IL-2).

Samples from 50 normal donors with no recorded previous exposure to PE38and with a broad distribution of human leukocyte antigen (HLA) alleleswere analyzed. The cells of this population contained naïve T cellepitope. Samples from nine mesothelioma and seven hairy cell leukemia(HCL) patients that were previously treated with PE-based RIT and whohad developed high levels of neutralizing antibodies were also analyzed.The cells of this population contained memory T cell epitopes. Theresponses of the naïve donors and the previously treated patients areshown in a heat map format in FIGS. 1A-1B.

FIG. 1A presents the positive and negative responses of the 50 naïvedonors. The responses are shown as a percentage of responsive spots foreach donor. The strongest positive response is shown as >20%, mediumresponses are shown as 10%-20%, weak responses are shown as 3%-10%, andthe absence of a response (negative) is shown as <3%. The resultsincluded a total of 258 positive responses, and 201 of those were weak(3-10%), 44 were medium (10-20%), and 13 were strong (>20%), leaving atotal of 5292 negative responses (<3%). There were 122 responses indomain II (peptides 1-38) including 11 strong, 24 medium, and 87 weakresponses. There were 136 responses in domain III (peptides 39-111)including two strong, 20 medium and 114 weak responses.

Peptides 15 and 14 (in pool 3) provided the strongest and most frequentresponses in the study with 21 and 18 positive responses, respectively.A total of 36 peptides did not provide any response. Sixteen of the 50naïve donors had a single epitope giving a response, as indicated byone, two, or three responses in overlapping consecutive peptides.Fifteen donors had two separate epitopes, 14 had three epitopes, andfive had four epitopes. On 22 occasions, a fine screen of a positivepool did not provide a specific peptide response or was not done,resulting in five consecutive positive responses in the heat map.

FIG. 1B illustrates the positive and negative responses of the ninemesothelioma and the seven HCL patients. It was found that themesothelioma patients provided 919 negative responses and 80 responses,including two strong responses (>20%), four medium responses (10-20%),and 74 weak responses (3-10%). The two strong responses were in peptides13 and 14, and the medium responses were in peptides 15, 35, 57 and 74.Thirty-five of the responses were in domain II (peptides 1-38) and 45 indomain III (peptides 38-111). The hot spot pattern created in the map issimilar to the hot spot pattern in the naïve donors map. Theresponsiveness (meaning how many epitopes were found for each patient)was higher than the donor's pattern with one patient having twoepitopes, four patients having three epitopes, two patients having fourepitopes and two more patients having five epitopes.

Moreover, it was found that the seven HCL patients provided three strongresponses (>20%), nine medium responses (10-20%), 37 weak responses(3-10%), and 728 negative responses (<3%). The strong responses were allin the consecutive peptides 75, 76 and 77. Twenty-two of the responseswere in domain II and 37 responses were in domain III. Interestingly,the immunodominant epitope in peptides 14 and 15 that was identified inmore than 40% of the naïve donors and 55% of the mesothelioma patients(five out of nine samples) did not have a single response in the HCLpatients. One patient responded to peptides 12 and 13 that have aminoacids in common with peptides 14 and 15. However, no responses werefound directly to peptides 14 and 15. This may be attributed to thebiased HLA distribution of those patients' samples or to the nature ofthe disease.

Overall, eight major epitopes were identified in PE38 that account for93% of the responses. Because all T cell epitopes in domain II could beeliminated using the LR deletion, the five epitopes remaining in domainIII were the focus of subsequent study. Table 1A summarizes theepitopes' locations, sequences, response rates, and the mutation thatdiminishes each with minimal disturbance to cytotoxic activity. Theepitope ranking was composed of a number of factors, including thenumber of naïve donors responding to the epitope, the relative strengthof the response, the number of mesothelioma patients that responded,and, to a lesser extent, the HCL patients' responses. The epitopes fromHCL patients were evaluated to a lesser extent in this analysis becauseof the HLA bias of the patient cohort and in the HCL patient population,in which HCL patients have an enrichment of BRB1_11 (Annino et al.,Leuk. Lymphoma, 14 (Suppl.) 1:63-5 (1994)). Epitope 2 spans fivepeptides. To simplify analysis, it was divided into two sub-epitopes:peptides 77-78 (2A) and peptides 75-76 (2B). Some donors responded toboth 2A and 2B epitopes, while others responded to only one. Epitope 6(peptides 93-96) and epitope 8 (peptides 56-59) were also divided intotwo sub-epitopes.

TABLE 1A Responses Meso- Relative Epitope Donors thelioma HCL cytotoxicranking Peptide # Sequence (n = 50) (n = 9) (n = 7) Mutations Activity*1 13-15 LVALYLAARLSWNQV   21  6 1 Domain II  100% SEQ ID NO: 189deletion 2 A 77-78 GALLRVYVPRSSLPG  14^(a)   3^(a)  6^(a) R505A  100%SEQ ID NO: 190 B 74-76 IRNGALLRVYVPRSS  10^(a)   6^(a)  5^(a) R494A21-36% SEQ ID NO: 191 3  8-9 RQPRGWEQLEQCGYP  9  3 3 Domain II  100%SEQ ID NO: 192 deletion 4  5-6 LPLETFTRHRQPRGW 10  2 0 Domain II  100%SEQ ID NO: 193 deletion 5 67-68 WRGFYIAGDPALAYG  8  2 2 L477H  100%SEQ ID NO: 194 6A + B 93-96 GPEEEGGRLETILGWPLA  8  1 2 L552E  100%SEQ ID NO: 195 7 51-52 TVERLLQAHRQLEER  5  1 0 R427A  100%SEQ ID NO: 196 8A + B 56-59 FVGYHGTFLEAAQSIVFG  5  5 4 F443A >100%SEQ ID NO: 197 * Activity for a single point mutation in HA22 RIT andevaluated in CA46 cell line. ^(a)Donors and patients that responded toepitope 2A overlap with the patients and donors that responded to 2B.

To compare the results from naïve donors to immunotoxin-treatedpatients, two patient cohorts that made neutralizing antibodies againstthe RIT were studied. The patients' DRB1 HLA alleles are shown in Table1B. The same epitopes identified in the donor cohort were also presentin the patient cohorts. One cohort was from mesothelioma patientstreated with SS1P (anti-mesothelin Fv fused to P38) (Chowdhury et al.,Nat. Biotechnol., 17: 568-572 (1999)); the other from leukemia patientstreated with moxetumomab pasudotox (MP), a RIT including PE38 fused toan anti-CD22 Fv (Kreitman et al., Clin. Cancer Res., 17: 6398-6405(2011)). The naïve donor epitope responses ranged from 1-4 epitopes perdonor, with an average of 2.1, and the patient responses ranged from 1-7per patient, with a higher average of 3.4 (P<0.001 in Student T test).This suggests that some responses in the naïve population were too weakto be detected by this method, and were amplified after exposure toimmunotoxin. The patient samples did not identify any major epitopesthat were not identified using the donor cohort.

TABLE 1B HLA haplotype Donor Diagnosis DRB1 71509WBp HCL 04, 11102609aph HCL 1103, 1303 112309aph HCL 0404, 0701 121809aph HCL 07, 11050710aph HCL 01, 07 021012baph HCL 04, 07 071912aph HCL 07, 11120909aph Mesothelioma 0410, 1501 011410aph Mesothelioma 0101, 0801012810aph Mesothelioma 0301, 1501 050510aph Mesothelioma 0401, 1302091510aph Mesothelioma 07, 15 022811aph Mesothelioma 1301, 1302100711aph Mesothelioma 0103, 03  021012aph Mesothelioma 0101, 0101031612aph Mesothelioma 0401, 12 

Example 2

This example demonstrates the elimination of T cell epitopes in domainIII.

For each one of the epitopes in domain III that are described in Table1, alanine scanning mutagenesis was performed. The alanine mutant wasincorporated into the RIT according to the following 11 steps: (1) alist was made of all alanine peptide variants for each epitope; (2) insilico prediction was performed to rule out alanine variants that hadincreased binding to at least six HLA alleles using an HLA bindingalgorithm (Immune Epitope Database, IEDB) (Wang et al., PLoS Comput.Biol., 4:e1000048 (2008)) that measured their ability to bind to 13major HLA groups; (3) alanine variants were assayed using 8-15 donorsand patient samples with in vitro expansion and ELIspot; (4) an alaninemutation with diminished T cell activation was identified; (5) thealanine mutation was cloned into the HA22-LR plasmid, and a MT wasconstructed with a single point mutation; (6) activity was studied usingthe WST8 assay.

If the protein was aggregated or has low cytotxic activity, (7) insilico prediction and crystal structure was used to identify alternativeamino acids to alanine and (8) the new mutation was cloned into anHA22-LR plasmid, and a MT was constructed with a single point mutationand activity was studied. If the protein was aggregated or had lowcytotoxic activity, then the next best alanine mutant was identified asin step 4 and step 5 was carried out. If the protein was active, step 10was carried out.

If the protein was active, (10) validation was carried out to determinewhether the epitope was diminished and that no new epitopes were createddue to the mutation; and (11) the successful point mutations werecombined into a single RIT.

The results for all epitopes are shown in Tables 2-7. In Tables 2-7,underlined values represent <10%, D=Donor, Pt=patient,“Meso”=mesothelioma, the position where the amino acid was replaced withalanine is underlined, and the average values are bolded. Alaninevariants were assayed using 8-15 donor and patient samples with in vitroexpansion and ELIspot.

TABLE 2 CD4+ T cell response to alanine variant peptides and WT76.donor/ HCL Meso HCL Meso Meso Mean patient Pt Pt Pt Pt Pt D1 D2 D3 D4 D5n = 16 No   3%   0%   0%   1%   0%   0%   9%   0%  17%   1%   0% peptideWT76 IRNGALLRVYVPR 100% 100% 100% 100% 100% 100% 100% 100% 100% 100%100% SS SEQ ID NO: 198 I493A ARNGALLRVYVPR   2%  13%   0%  36%  52%   6% 20% 110%  71%  76%  25% SS SEQ ID NO: 199 R494A IANGALLRVYVPR   1%   0%  2%   2%   0%  12%  26% 111%  88%  94%  24% SS SEQ ID NO: 200 N495AIRAGALLRVYVPR  95%  25%   2%  31%  12%  35%  40% 102%  72%  72%  34% SSSEQ ID NO: 201 G496A IRNAALLRVYVPR 107%  58%  18%  63%  34%  18%  37% 96%  44%  99%  42% SS SEQ ID NO: 202 L498A IRNGAALRVYVPR   4%   4%   0%  4%  27%   0%  80%   6%  16%  31%   5% SS SEQ ID NO: 203 L499AIRNGALARVYVPR   7%   0%   0%   1%   2%   6%  31%  30%  15%  57%   3% SSSEQ ID NO: 204 R500A IRNGALLAVYVPR   5%   4%  24%   2%   0%  24%  26%  1%  20%   6%   4% SS SEQ ID NO: 205 V501A IRNGALLRAYVPR   5%   0%   7% 13%  29%   0%  46%  25%  27%  48%  11% SS SEQ ID NO: 206 Y502AIRNGALLRVAVPR  29%   4%  18%  23%  70%  12%  23%   5%  20%   0%  19% SSSEQ ID NO: 207 V503A IRNGALLRVYAPR  54%  58%  47%  68%  30%  41%  63% 35%  27%  43%  41% SS SEQ ID NO: 208 P504A IRNGALLRVYVAR  91% 125%  49%122% 117% 206%  60%  35%  26%  18%  74% SS SEQ ID NO: 209 R505AIRNGALLRVYVPA  95%  83%  87% 127% 133% 147% 131%  36%  23%  30%  87% SSSEQ ID NO: 210 S506A IRNGALLRVYVPR  92% 138%  44% 107% 100% 312% 214%101%  55%  82% 106% AS SEQ ID NO: 211 S507A IRNGALLRVYVPR  99%  96%  51% 69% 106% 235% 137%  71%  83%  84%  94% SA SEQ ID NO: 212

TABLE 3 T cell response to alanine variant peptides and WT77. D/ HCL HCLHCL HCL Meso Mean Pt Pt 1 Pt 2 Pt 3 Pt 4 Pt 1 D1 D2 D3 (n = 8) No   0%  0%   0%  12%   1%  18%   9%   7%   6% peptide wt 77 GALLRVYVPRSSLPG100% 100% 100% 100% 100% 100% 100% 100% 100% SEQ ID NO: 213 G496AAALLRVYVPRSSLPG  29%  51% 126% 151%  37%  58% 112% 123%  86%SEQ ID NO: 214 A497G GGLLRVYVPRSSLPG 103%  68% 102% 167%  71% 108% 129%126% 109% SEQ ID NO: 215 L498A GAALRVYVPRSSLPG  61%  38%   7% 166%  43% 20%  13% 140%  61% SEQ ID NO: 216 L499A GALARVYVPRSSLPG  46%  24%  33%162%   1%  26%  73%  61%  53% SEQ ID NO: 217 R500A GALLAVYVPRSSLPG  59% 31%   1% 189%   6%  16%   1% 124%  53% SEQ ID NO: 218 V501AGALLRAYVPRSSLPG   7%  18%  18%  84%  15%  26%  49%  28%  31%SEQ ID NO: 219 Y502A GALLRVAVPRSSLPG  14%   1%   4%  10%   0%  25%   8%  9%   9% SEQ ID NO: 220 V503A GALLRVYAPRSSLPG  17%   1%  15%  18%   1% 39%  49%   9%  19% SEQ ID NO: 221 P504A GALLRVYVARSSLPG  46%  32%  38%127%  36%  43%  12%  41%  47% SEQ ID NO: 222 R505A GALLRVYVPASSLPG   7%  1%  28%  15%   2%  24%  39%  22%  17% SEQ ID NO: 223 S506AGALLRVYVPRASLPG  68%  42% 107% 128%  48%  93% 122%  50%  82%SEQ ID NO: 224 S507A GALLRVYVPRSALPG  35%  37%  86% 164%  60% 106%  96%224% 101% SEQ ID NO: 225 L508A GALLRVYVPRSSAPG  8%   0%  73%  12%  46% 99%  88%  15%  43% SEQ ID NO: 226 P509A GALLRVYVPRSSLAG 10%   4%  87% 57%  72% 101% 119%  16%  58% SEQ ID NO: 227

TABLE 4 T cell response to alanine variant peptides and WT 67 % from HCLHCL HCL Meso Meso Mean WT Pt1 Pa 2 Pt 3 Pt1 Pt 2 D1 D2 D3 D4 D5 (n = 14)No   6%   3%   3%  13%   1%   1%  13%   0%   0%   0%  13% peptide WT 67WRGFYIAGD 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% PALAYGSEQ ID NO: 228 F469A WRGAYIAGD  80%  68%  93%  11%  81% 104%  35%  97% 88%  60%  69% PALAYG SEQ ID NO: 229 Y470A WRGFAIAGD  61%   6%  61%   6% 68%  48%  67%  29%  16%  92%  43% PALAYG SEQ ID NO: 230 I471A WRGFYAAGD 17%  36%  22%   7%  35%  20%  20%  22%  12%  55%  24% PALAYG SEQ ID NO:231 A472G WRGFYIGGD  37%  81%  38%  20%  60%  84%  75%  46%  19%  76% 48% PALAYG SEQ ID NO: 232 P475A WRGFYIAGD  72%  18%  46%  73%  38%  34% 57%  15%  16%  70%  43% AALAYG SEQ ID NO: 233 A476G WRGFYIAGD  75%  49% 40% 110%  32% 207%  26%  67%  35% 115%  70% PGLAYG SEQ ID NO: 234 L477AWRGFYIAGD  54%  32%  54%  92%  52%  25%  42%  26%  16%  71%  44% PAAAYGSEQ ID NO: 235 A478G WRGFYIAGD  80%  81%  62%  96%  58% 134%  24%  63% 98%  66%  72% PALGYG SEQ ID NO: 236 Y479A WRGFYIAGD 114% 106%  76% 250%138% 108% 117%  71%  21%  94% 104% PALAAG SEQ ID NO: 237

TABLE 5Alanine scanning for peptides 93-94 GPEEEGGRLETILGWPLA (SEQ ID NO: 238)HCL HCL Meso Meso Meso Mean Pt 1 D1 D2 Pt 2 Pt1 Pt 2 Pt 3 D3 D4 D5 n =10 Responder groups 93 93 93 94 94 94 94 94 94 94 No   3%   9%   2%   7%  4%   3%  15%  35%  25%  12%  11% peptide WT 93- GPEEEGGRLETILGWPLA100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 94 SEQ ID NO: 238E547A GPEAEGGRLETILGWPLA  73%  18% 254%  85% 120% 122% 136%  73% 106% 90% 108% SEQ ID NO: 239 E548A GPEEAGGRLETILGWPLA  58%  24% 170% 112% 98%  86% 137% 102% 207%  87% 108% SEQ ID NO: 240 R551AGPEEEGGALETILGWPLA  62%   6%   2%  95%  70%  42%  54%  40%  67%  49% 49% SEQ ID NO:241 L552A GPEEEGGRAETILGWPLA  54%  44%  10%  91%  35% 11%  14%  85%  51%  46%  44% SEQ ID NO: 242 T554A GPEEEGGRLEAILGWPLA 77%   9%   5%  94% 133% 130% 129% 110% 95% 112%  89% SEQ ID NO: 243I555A GPEEEGGRLETALGWPLA 111% 176% 119%  67%  25%  15%  36%  63%  36% 36%  69% SEQ ID NO: 244 L556A GPEEEGGRLETIAGWPLA  24% 235%   3%  40% 10%  13%  43%  35%  83%  29%  51% SEQ ID NO: 245 W558AGPEEEGGRLETILGAPLA  20% 200%  13%  13%  26%  7%  26%  46%  54%  31%  44%SEQ ID NO: 246 P559A GPEEEGGRLETILGWALA  24% 197% 208%  95%  69%  37% 74%  65% 121%  46%  94% SEQ ID NO: 247 L560A GPEEEGGRLETILGWPAA  81%321% 162%  89% 132% 117%  75%  46% 138%  95% 126% SEQ ID NO: 248

TABLE 6 Alanine scanning for peptide TVERLLQAHRQLEER (SEQ ID NO: 249)HCL Meso Meso Meso Mean Pt 1 Pt 1 Pt 2 Pt 3 D1 D2 D3 n = 7 No   0%   1%  0%   1%  13%  14%   7%   5% peptide WT51 TVERLLQAHRQLEER 100% 100%100% 100% 100% 100% 100% 100% SEQ ID NO: 249 R421A TVEALLQAHRQLEER  23% 87%   2%   5%  22%  12%  26%  25% SEQ ID NO: 250 L422A TVERALQAHRQLEER 29%  18%   0%   1%  31%  10%  16%  17% SEQ ID NO: 251 L423ATVERLAQAHRQLEER  47%  21%  11%   3%   8%  10%   6%  14% SEQ ID NO:252A425G TVERLLQGHRQLEER 105%  51%   4%   6%  57%  16%  68%  52%SEQ ID NO: 253 R427A TVERLLQAHAQLEER  78%  81%   1%   2%  37%  10%  35% 37% SEQ ID NO: 254 L429A TVERLLQAHRQAEER  63%  64%  38%  36%  28%  19%124%  67% SEQ ID NO: 255 E430A TVERLLQAHRQLAER 100% 100% 242%  73% 112% 26%  99% 108% SEQ ID NO: 256 R432A TVERLLQAHRQLEEA 105%  92%  87%  69% 65%  57% 142% 106% SEQ ID NO: 257

TABLE 7Alanine scanning for 18 mer peptide FVGYHGTFLEAAQSIVFG (57-58) (SEQ ID NO: 258)HCL Meso Meso HCL HCL Meso Meso Pt1 Pt1 Pt 2 D1 Pt 2 Pt 3 Pt 3 Pt 4 D2D3 Avg Responder groups 57 57 57 57 58 58 58 58 58 58 No   0%   0%   0%  4%   0%   1%   1%   0%  10%   9%   4% peptide WT 57-FVGYHGTFLEAAQSIVFG 100% 100% 100% 100% 100% 100% 100% 100% 100% 100%100% 58 SEQ ID NO: 258 F436A AVGYHGTFLEAAQSIVFG  57%  98% 159%   7% 103%103%  89%  45%  41% 123%  83% SEQ ID NO: 259 V437A FAGYHGTFLEAAQSIVFG 84%  78% 130%  81%  97%  95%  96%  85% 102% 148%  99% SEQ ID NO: 260G438A FVAYHGTFLEAAQSIVFG  67%  52%  78%  79%  71%  88% 118%  91%  98%  68%  83% SEQ ID NO: 261 Y439A FVGAHGTFLEAAQSIVFG  22%  96%  21%  17% 79%  97% 125% 147% 110% 154%  91% SEQ ID NO: 262 H440AFVGYAGTFLEAAQSIVFG  11%  12%  95%   6%  46% 113% 108% 120%  90% 102% 75% SEQ ID NO: 263 T442A FVGYHGAFLEAAQSIVFG  77%  84% 151%  31%  48%102%  93%  50%  59% 127%  82% SEQ ID NO: 264 F443A FVGYHGTALEAAQSIVFG  2%   0%  40%   7%   5%   2%   1%   1%  20%   7%  10% SEQ ID NO: 265L444A FVGYHGTFAEAAQSIVFG  69%  54%  74% 140%   9%   4%  55%   8%  14%  0%  37% SEQ ID NO: 266 A446G FVGYHGTFLEGAQSIVFG  80%  40%  47% 119% 80%  37% 107% 125%  69%  16%  78% SEQ ID NO: 267 A447GFVGYHGTFLEAGQSIVFG  65% 104%  57% 103%  19%  28% 118%  87%  43%   7% 67% SEQ ID NO: 268 S449A FVGYHGTFLEAAQAIVFG  98% 126%  93% 104%  23% 65% 128% 123% 163% 200% 110% SEQ ID NO: 269 I450A FVGYHGTFLEAAQSAVFG138% 159% 130% 124%   2%  66% 108% 104%  31%  11%  78% SEQ ID NO: 270V451A FVGYHGTFLEAAQSIAFG 119% 127% 162% 121%  82% 117% 142% 118% 137% 50% 116% SEQ ID NO: 271 F452A FVGYHGTFLEAAQSIVAG 119% 126% 154% 156% 99% 104% 103% 151% 104% 123% 127% SEQ ID NO: 272

Epitopes 2A and 2B were scanned separately to cover the 9 mer core ofall five peptides that gave responses. Y502A diminished the responses ofboth epitopes (Tables 2 and 3). For epitopes 5 and 7, alanine mutantswere compared to peptide 67 and 51, respectively, and I471A and L423Ahad the lowest T cell response (Tables 4 and 6). To cover all 9 mercores in epitopes 6 and 8 that contained four positive peptides, 18 merWT and alanine variants were synthesized. It was found that L552A wasthe most effective in lowering the response in epitope 6 (Table 5) andF443A was best for epitope 8 (Table 7).

Example 3

This example demonstrates the cytotoxic activity of point mutationproteins in HA22-LR.

Step 4 of Example 2 was carried out. The best variants identified in thealanine scan or using further in silico prediction are described inTable 1A.

Forty mutant RITs were constructed. The cytotoxic activity of pointmutation proteins in HA22-LR was evaluated with respect to CA46, Raji,and Daudi cells. Yields of purified protein, calculated accessiblesurface areas (Lee et al., J. Mol. Biol., 55:379-400 (1971); Roscoe etal., Infect. Immunity, 62:5055-5065 (1994)) of WT amino acids residuesand cytotoxic activity of each RIT are shown in Table 8.

TABLE 8 Protein Protein activity Muta- ASA yield Mean relative activity(%) ± SD Peptide tion (Å²)* (mg) CA46 Raji Daudi 76 R500A 0 A 76 L498A 4A 76 L499A 0.1 A 76 R494A 35 2.0 21 ± 3 33 ± 10 36 ± 8  76 R494D 35 3.2311 77 R505A 150 4  78 ± 13 185 ± 106 76/77 Y502A 0 A 76/77 Y502H 0 A76/77 Y502F 0 A 76/77 Y502K 0 A 67 I471A 4 1.6 24 67 I471V 4 4 83 108 67I471M 4 3 3 67 I471H 4 A 67 I471G 4 A 67 I471S 4 1.3 2 67 I471D 4 A 67Y470A 98 2.3 <1 <1 67 L477A 7 1.4  54 ± 21 47 ± 13 67 A476G 38 5 67 ± 484 67 L477G 7 4 14 ± 6 14 ± 6  67 L477T 7 2.5 <1 <1 67 L477H 7 3 101 ±22 95 ± 13 51 L422A 0 2 <1 51 L423A 22 4 30 51 L423N 22 3 5 51 L423T 223.2 50 76 51 L423S 22 5 65 51 R421A 16 1 <10 51 R427A 142 0.71 185 ± 85150 ± 61  150 ± 4  93 R551A 100 4 208 188 ± 464 94 W558A 40 1 <1 >2 94W558D 40 1 <1 94 W558N 40 0.7 <1 94 L556A 0 1.2 2 0   2 ± 0.4 94 L556V 04.2  95 ± 32 77 94 L552N 82 2.4 200 94 L552E 82 2.1 110 57/58 F443A 123.6  174 ± 110 113 ± 60  57/58 F443V 12 2.26 95 A—aggregated *AccessibleSurface Areas (ASA) were calculated for the wild type residue.

Example 4

This example demonstrates that the substitutions in PE domain IIIdiminish the epitope and do not create new epitopes due to the mutation.

The response of three donor samples and one patient sample to 22 peptidepools after stimulation with either HA22 (wt) or LR-R494A was measured.“LR” denotes the deletion of all of domain II except the furin cleavagesequence. The results are shown in FIGS. 2A-2D. As shown in FIGS. 2A-2D,the epitope in peptide 76 was diminished by the R494A mutation.

The response of two donor samples and one patient sample to 22 peptidepools after stimulation with either HA22 (wt) or LR-R505A was measured.The results are shown in FIGS. 3A-3C. As shown in FIGS. 3A-3C, theepitope in peptide 77 was diminished by the R505A mutation.

The response of two donors to 22 peptide pools after stimulation witheither HA22-LR (WT) or HA22-R551A and restimulation with the appropriatepeptides was also measured. The results are shown in FIGS. 4A-4B. Asshown in FIGS. 4A and 4B, the mutation R551A diminished the epitope inpeptide 93.

The response of cells from two donors and two patients after stimulationwith RIT and restimulation with peptides 93, 94 with either the WT aminoacid sequence, L552E or L552N was measured. The results are shown inFIGS. 5A-5D. As shown in FIGS. 5A-5D, the epitope in peptides 93 and 94was diminished by the L552N and L552E mutations.

The response of three donors to 22 peptide pools after stimulation witheither HA22-LR (WT) or LR-R427A and restimulation with the appropriatepeptides was also measured. The results are shown in FIGS. 6A-6C. Asshown in FIGS. 6A-6C, the epitope in peptide 51 was diminished by theR427A mutation.

The response of two patients and one donor to pools 8-22 afterstimulation with either HA22-LR (WT) or LR-F443A and restimulation withthe appropriate peptides was measured. The results are shown in FIGS.7A-7C. As shown in FIGS. 7A-7C, the epitope in pools 11 and 12 wasdiminished by the F443A mutation.

Four donor samples were stimulated with RIT and restimulated withpeptide 67 containing a valine or alanine mutation at position 1471. Theresponse is shown in FIGS. 9A-9D in Spot Forming Cells (SFC)/10⁶ cells.As shown in FIGS. 9A-9D, mutation I471V did not diminish the epitope inpeptide 67.

T cell activation as a response to stimulation with peptide 67 or 68that contained an alanine or histidine mutation at position L477 or nomutation (WT) was also measured. The results (SFC/10⁶ cells) are shownin Table 9 and FIGS. 10A-10D. As shown in FIGS. 10A-10D and Table 9, themutation L477H diminished the epitope in peptide 67 and 68.

TABLE 9 hcl meso 11410 41311 102909 021012 031612 Mean WT 67 100 100 100100 100 100 67 L477A 81 21 24 133 39 60 67 L477H 0 30 123 65 23 48 WT 68100 100 100 100 100 100 68 L477A 69 19 31 62 38 44 68 L477H 2 34 46 3248 32

Cells from two patients were stimulated with RIT and restimulated withpeptide 93, 94 or 95 with either the WT amino acid sequence or L556V.The results are shown in FIGS. 11A and 11B. Mutation L477V did notdiminish the epitope in peptide 94.

Example 5

This example demonstrates the activity of combinations of mutations inHA22-LR.

To combine the point mutations that were effective in diminishing the Tcell epitopes and maintain good cytotoxic activity, genes were designedthat contain different combinations of point mutations. The genes werecloned into the HA22-LR RIT plasmid. Table 10 shows the seven RITs thatwere constructed and also HA22-LR WT. Table 10 also shows the cytotoxicactivity in two cell lines and the calculated remaining T cell response.The calculated remaining response is the sum of the responses shown inthe epitope heat map for each of the epitopes, assuming that themutation will eliminate the responses in the epitope completely. Thiscalculation does not take into account the strength of the response.

As shown in Table 10, out of the 258 responses identified in the donorscreen of PE38, 136 responses were in domain III and are present inHA22-LR. Elimination of the epitopes in peptide (77-78), (51-52) and(57-58) with mutations R505A, R427A and F443A (HA22-LR-T2) reduced thecalculated responses to 82 responses and maintained a very activeprotein with 280% and 135% relative activity in CA46 and Raji cells,respectively, compared to WT. HA22-LR-T3 contains four mutations: R505A,R427A, F443A and R494A and had diminished activity of 55% and 50%.HA22-LR-T7 also has four mutations, (R505A, R427A, F443A and R551A) andhad good cytotoxic activity with 113% and 100% relative activity in CA46and Raji cells. For the construction of HA22-LR-T5, five mutations,R505A, R494A, L427A, R551A and F443A, were inserted which reduced thecalculated remaining response to 39 responses. However, the protein'scytotoxic activity was severely reduced to 24% and 19% relative activitywith EC₅₀>1 ng/ml. HA22-LR-T9 also had five point mutations (R505A,R427A, F443A, R494A and L477A). T9 had 46 calculated remainingresponses; all 46 remaining responses were weak or medium). Itmaintained cytotoxic activity with EC₅₀<1 ng/ml with relative activityof 36% and 26% in CA46 and Raji cells, respectively. HA22-LR-T11substitutes L477A in T9 with L477H which improved that activity to 0.2ng/ml with 85% relative activity in CA46. Lastly, T18 (also referred toas “LMB-T18”) was the first RIT that contains six mutations anddiminished all the major epitopes that were identified. It had goodrelative activity with IC50<0.3 ng/ml and relative activity of 63% and104% compared to HA22-LR in CA46 and Raji cells. To improve cytotoxicactivity, a Gly-Gly-Ser peptide linker was inserted after the furincleavage site.

TABLE 10 T11- T2-HA T3-HA T5-HA T7-HA T9-HA HA T18 HA22- HA22- HA22-HA22- HA22- HA22- HA22- HA22- LR LR LR LR LR LR LR LR RIT backbone R505A77-78 + + + + + + + R494A 75-76 + + + + + L477A 67-68 + L477H 67-68 + +R427A 51-52 + + + + + + + R551A 93-94 + + L552E + F443A57-58 + + + + + + + Cytotoxic Activity CA46 IC50* 0.17 0.06 0.31 0.710.15 0.48 0.2 0.27 (ng/ml) CA46 Relative 100% 280% 55% 24% 113% 36% 85% 63% activity Raji IC50* 0.23 0.17 0.46 1.18 0.23 0.88 0.24 (ng/ml) RajiRelative 100% 135% 50% 19% 100% 26% 104% activity Calculated 136/25882/258 62/258 39/258 59/258 46/258 46/258 19/258 Responses *IC50 valuesare an average of 2-9 assays, depending on the protein.

To characterize the cytotoxic activity properties of T3 and T18, WST-8assays were performed in four cell lines that express CD22: ca46, Raji,Daudi and HAL-01. Each assay was performed three times. Table 11A showsthe average EC₅₀ values for each cell line. Both HA22-LR-T3 andHA22-LR-T18 were very cytotoxic, although somewhat less than the parentmolecule HA22-LR-GGS. Compared with HA22-LR-GGS, HA22-LR-T18 showedrelative activity that ranged between 28%-55% with IC₅₀<1 ng/ml in thefour cell lines. HA22-LR-T3 was also very active, however, less activethan T18.

TABLE 11A Summary of EC₅₀ and relative activity of HA22-LR-T3 andHA22-LR-T18 RITs HA22-LR- HA22-LR- HA22-LR- Cell Line GGS T3 T18 CA46EC₅₀± 0.1 0.4 0.3 (ng/mL) SD ±0.003 ±0.08 ±0.01 Relative 100 18 28activity (%) Raji EC₅₀± 0.1 0.3 0.2 (ng/mL) SD ±0.04 ±0.15 ±0.07Relative 100 34 55 activity (%) Daudi EC₅₀± 0.1 0.4 0.3 (ng/mL) SD ±0.040.08 ±0.17 Relative 100 31 38 activity (%) HAL-01 EC₅₀± 0.3 1.5 0.7(ng/mL) SD ±0.05 ±0.47 ±0.16 Relative 100 20 42 activity (%)

Cytotoxicity assays of LMB-T18 were performed on four CD22 expressingcell lines and compared with the cytotoxic activity of MP (FIG. 27A,Table 11B). LMB-T18 was very potent with an EC50<10 pM in all celllines. Compared to MP, LMB-T18 had a small increase of 53% in activityin CA46 cells, 54% in Daudi cells and >200% in HAL-01 cells (p=0.2, 0.06and 0.01 respectively in Student T test); however, in Raji cells LMB-T18had a 52% activity decrease (p=0.3 in Student T test). Without beingbound by a particular theory or mechanism, it is believed that thedecrease in activity in Raji cells was probably due to the domain IIdeletion (Weldon et al., Blood, 113: 3792-3800 (2009)). The stability ofLMB-T18 was compared with MP by heating samples for 15 minutes atvarious temperatures and, after cooling, measuring residual cytotoxicactivities (FIG. 27B) on Raji cells. MP lost 50% of its activity after15 minutes incubation at 56° C. Unexpectedly, LMB-T18 was moreheat-resistant (p<0.05 in Student T test); it only lost 50% of itsactivity after a 15-minute incubation at 70° C. Cells from seven HCL andsix chronic lymphocytic leukemia (CLL) patients were used to determineactivity on patient cells. FIGS. 27C-27D show that LMB-T18 is moreactive than MP on CLL cells though this difference is not seen with HCLcells.

TABLE 11B EC50 ± SD (pM) Relative Cell line MP LMB-T18 activity (%) Pvalue CA46 (n = 4) 3.4 ± 1.7 2.2 ± 0.4 153 0.2 Raji (n = 4) 1.1 ± 0.32.3 ± 0.6 48 0.3 Daudi (n = 4) 3.7 ± 1.6 2.4 ± 0.8 154 0.06 HAL-01 (n =4) 25.7 ± 3   8.1 ± 1  318 0.01

Example 6

This example demonstrates the relative activity of B and T cell epitopemodified RIT.

B cell epitopes in PE38 were previously identified and the bulky surfaceresidues were mutated with alanine or serine to reduce antigenicity (Liuet al., Protein Eng. Dec. Sel., (25)(1): 1-6 (2012)). The previouslyidentified mutant RIT with low antigenicity was named SS1LO10R and itcontained the following mutations: R505A, R427A, R490A, R463A, R467A,R538A.

To make a minimally immunogenic RIT targeting mesothelin, the LO10modifications were incorporated into SS1P and then the T cellepitope-point mutations were incorporated one by one. Cytotoxic activitywas tested for each one of the variants using mesothelin expressing celllines (A9/431 and HAY). Two mutations R505A and R427A were found to beeffective in diminishing both B and T cell epitopes.

Table 12 summarizes the cytotoxic activity of the mutated PE in two celllines and the calculated remaining response of the combination RIT thatwere constructed. Combination of SS1-LO10 with a single point mutationF443A (SS1-LO10-T2) had high (100%-115%) relative activity thatcorresponded to the activity of these point mutations in HA22-LR.Combination of R494A and R551A in SS1-LO10-T1 gave a very low activityindicating that the combination of R551A and R494A diminished proteinactivity in both HA22 and SS1-LO10.

TABLE 12 Relative activity of B and T cell epitope modified RIT RITBackbone R505A R494A L477A R427A R551A 77-78 75-76 67-68 L477H 51-5293-94 L552E L552N SS1 LO10R SS1 SS1 + + + + LO10R- LO10R T1 SS1 SS1 + +LO10R- LO10R T2 SS1 SS1 + + + LO10R- LO10R T3 SS1 SS1 + + + LO10R- LO10RT4 SS1 SS1 + + + LO10R- LO10R T7 SS1 SS1 + + + LO10R- LO10R T8 SS1SS1 + + + + LO10R- LO10R T9 SS1 SS1 + + + + LO10R- LO10R T11 SS1SS1 + + + + + LO10R- LO10R T14 SS1 SS1 + + + + + LO10R- LO10R T15 RITCytotoxic activity Backbone A431/H9 HAY F443A Relative RelativeCalculated 57-58 R456A IC50* (ng/m) activity IC50* (ng/m) activityresponses SS1 0.3 0.5 136/258  LO10R SS1 1.6 19% 50 10% 52/258 LO10R- T1SS1 + 0.17 176% 0.5 100% 82/258 LO10R- T2 SS1 + 0.29 103% 0.6 83% 62/258LO10R- T3 SS1 + + 2 15% nd 62/258 LO10R- T4 SS1 + 0.6 50% 2 50% 59/258LO10R- T7 SS1 + + 0.9 33% 2.5 20% 59/258 LO10R- T8 SS1 + 1.1 27% 6.9 7%46/258 LO10R- T9 SS1 + 0.42 71% 7 7% 46/258 LO10R- T11 SS1 + 0.23 130%1.5 33% 19/258 LO10R- T14 SS1 + 0.27 111% 1.9 26% 19/258 LO10R- T15*IC50 values are an average of 1-9 assays, depending on the protein;nd—not done

SS1 LO10R-T11, which contained R505A, R427A, R494A, F443A and L477H, had71% activity in H9 cells but only 7% in HAY cells (assay in HAY was doneonce). T14 and T15, which contained R505A, R427A, R494A, F443A andL477H, with an additional mutation (L552E and L552N, respectively)had >100% relative activity in H9 cells and IC50 >1 ng/ml. These twomutants represent the maximal T cell deimmunization done in this projectthus far.

Mutant T14 was selected as the best candidate for clinical developmentbecause it includes the removal of B cell and many T cell epitopes, yetprovides good cytotoxic activity on mesothelin expressing cell lines.

Example 7

This example demonstrates the anti-tumor activity of T14.

Anti-tumor experiments were carried out by injecting saline (control) orT14 into nude mice with A431/H9 xenografts. Tumor size was measuredevery other day and reported in mm. Animals were weighed at the sametime and the weight was reported in grams. The results are shown inTable 13 below. In Table 13, the arrows show the day immunotoxin wasinjected. As shown in Table 13, doses up to 7 mg/kg×3 were welltolerated with no signs of toxicity in the mice. In addition, majortumor regressions were seen at all dose levels of T14.

TABLE 13 Days Group Mouse # Day 5 ↓ 7 ↓ 9 ↓ 12 ↓ 14 ↓ A 1 108 23 g 11422 g 74 23 g 46 36 T14 2 100 22 g 100 22 g 56 22 g 40 64 5 mg mg/kg × 5iv QOD 3  97 20 g  92 20 g 31 20 g 0 10 (100 ug/mouse) 4 101 19 g  94 19g 60 20 g 52 74 5  97 22 g  83 22 g 57 22 g 41 70 6 103 21 g  95 21 g 5821 g 37 58 Mean 101  96 56 36 52 B 1 100 22 g  91 20 g 81 20 g 86 78 T142  96 23 g 103 23 g 50 23 g 14 66 6 mg/kg × 3 iv QOD 3  10021 g 112 21 g91 22 g 54 88 (120 ug/mouse) 4 103 24 g 117 24 g 53 24 g 55 98 5 102 21g 121 20 g 62 20 g 82 125 Mean 100 109 67 58 91 C 1 100 21 g  98 21 g 3622 g 53 22 g 69 7 mg/kg × 3 iv GOD 2 101 20 g 126 19 g 44 20 g 65 21 g103 (140 ug/mouse) Mean 101 112 40 59 86 D 1 100 239 440 816 Control 295 133 234 612 saline 3 104 252 463 813 4 92 138 251 543 5 100 152 231589 Mean 98 183 324 675 Over 800

Example 8

This example demonstrates the thermal stability of T18.

The stability of T18 was investigated by heating the RIT for 15 minutesat various temperatures. Cytotoxic assays were subsequently performed.EC50 of the different heated proteins were calculated. The results areshown in FIG. 8. The fold increase in EC50 is shown. As shown in FIG. 8,it was found that T18 maintained cytotoxic activity in 60° C. and lost50% of its activity at 68° C. Leaver et al., Methods in Enzymology, 487:545-74 (2011).

Example 9.1

This example demonstrates the construction, expression, and purificationof anti-mesothelin deimmunized PE chimeric molecules with an elongatedlinker between the anti-mesothelin-Fab fragment heavy chain and the PE24 domain and the removal of predicted T cell epitopes in the linker.

The linker sequence used in WO 2012/154530 and Weldon et al., Mol.Cancer Ther., 12: 48-57 (2013) to fuse the dsFv fragment (via theC-terminus of VH domain) to the deimmunized PE was also used to fuse aFab fragment (via the C-terminus of CH1 domain) to the deimmunized PE.The resulting peptide region, including parts of the CH1 domain, thelinker, and part of the the deimmunized PE (SEQ ID NO: 273) was examinedby TEPITOPE analysis (Bian et al., Methods, 34: 468-475 (2004)).Potential T cell epitopes relevant for European (EU) and United Statespopulations were identified within the region between the CH1 domain andthe linker and the region between the linker and the PE24 domain.Potential T cell epitopes were characterized by a confidence number of 3or lower. The results are shown in Table 14. As shown in Table 14, thelinker region without the specific elongations (SEQ ID NO: 273) showedseven potential T-cell epitopes. One potential T-cell epitope waslocated in the transition region WEQLGGSPT (SEQ ID NO: 274), and theother six T-cell epitopes were located in the transition regionVEPKSCKAS (SEQ ID NO: 275). Both of these regions are relevant mainlyfor European (EU) and Asian populations.

The potential T cell epitopes were destroyed by inserting an amino acidsequence (insert) in each of the transition regions, thereby providingan insert on each of both sides of the furin cleavage site. The sequenceDKTH (SEQ ID NO: 276) was inserted into the linker at a location notdirectly adjacent to the furin cleavage sequence (FCS), and the sequenceGGG (SEQ ID NO: 277) was inserted into the linker at a location directlyadjacent to the FCS without potential T-cell epitopes. The resultingelongated peptide region, including a) parts of the CH1 domain, b) thelinker elongated at the Fab fusion area and elongated at the PE fusionarea, and c) part of the of the the deimmunized PE comprised amino acidSEQ ID NO: 278. The elongated peptide region was studied using TEPITOPEanalysis, and the results are shown in Table 15A. As shown in Table 15A,the elongated linker did not generate an alert in the TEPITOPE analysis(confidence numbers of 6 or higher) and, at the same time, providedimproved stability (see Example 9.2 below).

TABLE 14 Start Pos Sequence Score Confidence Profile Anchor InhibitorAllele 26  WEQLGGSPT 0.3 3 ********* 0 0 HLA-DRB1*0101 (SEQ ID NO: 274)8 VEPKSCKAS 1.6 2 ***A***** 1 0 HLA-DRB1*0802 (SEQ ID NO: 275) 8VEPKSCKAS 2.6 2 ***A***** 1 0 HLA-DRB1*0804 (SEQ ID NO: 275) 8 VEPKSCKAS2.6 3 ***A***** 1 0 HLA-DRB1*0806 (SEQ ID NO: 275) 8 VEPKSCKAS 1.9 3********* 0 0 HLA-DRB1*1102 (SEQ ID NO: 275) 8 VEPKSCKAS 1.9 3 *********0 0 HLA-DRB1*1121 (SEQ ID NO: 275) 8 VEPKSCKAS 1.9 3 ********* 0 0HLA-DRB1*1322 (SEQ ID NO: 275) Start f f f max Pos Sequence Pivot Pept.f (EU) f (US) (NE Asia) (SE Asia) (SW Asia) (f (all)) 26  WEQLGGSPT 1 08.0% 0.6% 5.7% 0.1% 4.1% 8.0% (SEQ ID NO: 274) 8 VEPKSCKAS 1 0 0.1%12.0%  2.6% 0.1% 0.8% 12.0%  (SEQ ID NO: 275) 8 VEPKSCKAS 1 0 0.1% 0.1%0.6% 0.0% 0.0% 0.6% (SEQ ID NO: 275) 8 VEPKSCKAS 1 0 0.1% 0.0% 0.1% 0.0%0.0% 0.1% (SEQ ID NO: 275) 8 VEPKSCKAS 1 0 0.2% 0.0% 0.0% 0.0% 1.0% 1.0%(SEQ ID NO: 275) 8 VEPKSCKAS 1 0 N.A. N.A. N.A. N.A. N.A. 0.0% (SEQ IDNO: 275) 8 VEPKSCKAS 1 0 N.A. N.A. N.A. N.A. N.A. 0.0% (SEQ ID NO: 275)all Alerts listed <=3

TABLE 15A Start Pos Sequence Score Confidence Profile Anchors InhibitorsAllele 30 WEQLGGGGG −1.2 9 ******I** 0 1 HLA-DRB1*0101 (SEQ ID NO: 279)33 LGGGGGSPT −0.7 9 ********* 0 0 HLA-DRB1*0102 (SEQ ID NO: 280) 30WEQLGGGGG(SEQ −1 9 ***A****I 1 1 HLA-DRB1*1307 ID NO: 279) 30WEQLGGGGG(SEQ 1.4 6 ********* 0 0 HLA-DRB5*0101 ID NO: 279) 30WEQLGGGGG(SEQ 1.4 6 ********* 0 0 HLA-DRB5*0105 ID NO: 279) Start PosSequence Pivot Peptide f (EU) f (US) f (NE Asia) f (SE Asia) f (SW Asia)30 WEQLGGGGG 1 0 8.0% 0.6% 5.7% 0.1% 4.1% (SEQ ID NO: 279) 33 LGGGGGSPT1 0 0.6% 0.1% 0.0% 0.0% 0.7% (SEQ ID NO: 280) 30 WEQLGGGGG(SEQ 1 0 0.0%0.0% 0.0% 0.0% 0.0% ID NO: 279) 30 WEQLGGGGG(SEQ 1 0 N.A. N.A. N.A. N.A.N.A. ID NO: 279) 30 WEQLGGGGG(SEQ 1 0 N.A. N.A. N.A. N.A. N.A. ID NO:279) no Alert <=3

Example 9.2

This example demonstrates the stability of the deimmunized PE fusionproteins and conjugates having the elongated linker of SEQ ID NO: 36.

Six Fab-Linker-PE24 variants were produced and compared with respect totheir thermal stability and propensity for aggregation. The six variantsall contained the same humanized Fab variant H1/L1 of the SS1 antibody,but differed with respect to the linker and the PE24 mutant. For thelinker, either the linker described in International Patent ApplicationPublication WO 2012/154530 (kasggrhrqprgweqlggs (SEQ ID NO: 281)) or theelongated linker with the additional amino acids (“long linker”; SEQ IDNO: 36 (dkthkasggrhrqprgweqlgggggs))) was used. As PE24 variants,LRO10R, LRO10R-456A (SEQ ID NO: 37) or LRO10R-456A-551A were employed inthe constructs.

FIGS. 12-13 show the level of aggregation of the two constructs with thePE24 variant LRO10R-456A that was measured in terms of the area underthe aggregate peak by size exclusion chromatography (SEC) afterincubation at 33° C. The two constructs in FIGS. 12-13 differed onlywith respect to the linker employed. The short linker SEQ ID NO: 281 wasused in construct cFP-0170 and the elongated linker SEQ ID NO: 36 wasused in construct cFP-0171.

FIGS. 14-16 show the thermal stability of constructs as measured bydynamic light scattering (DLS). For dynamic light scattering, sampleswere prepared at a concentration of 1 mg/mL in 20 mM histidine/histidinechloride, 140 mM NaCl, pH 6.0, transferred into an optical 384-wellplate by centrifugation through a 0.4 μm filter plate and covered withparaffin oil. The hydrodynamic radius was measured in a WYATT DYNAPROPlate Reader II repeatedly by dynamic light scattering while the sampleswere heated at a rate of 0.05° C./min from 25° C. to 80° C. Theaggregation onset temperature was defined as the temperature at whichthe hydrodynamic radius starts to increase. The results are shown inFIGS. 14-16. The DLS curves for the six constructs showed higheraggregation temperatures for the elongated linker compared to the shortpublished linker, with a strong shift to a higher aggregationtemperature for the LRO10R variant (FIGS. 14-16).

Table 15B shows the thermal stability of constructs cFP-0170 andcFP-0171 as measured by differential scanning calorimetry (DSC).Differential scanning calorimetry was performed using a MicroCal DSCsystem (GE Healthcare). The samples were adjusted to a proteinconcentration of approximately 1 mg/ml in 20 mM histidine/HCl, 140 mMsodium chloride, pH 6.0. The reference cell was filled with a buffercorresponding to the sample buffer. The samples were placed in thesample cell and heated from 4° C. to 110° C. at a heating rate of 60°C./hour. The pre-scan was 15 minutes, the filtering period was 10seconds, and the feedback mode/gain was set to passive. The midpoint ofa thermal transition temperature (Tm, or thermal transition temperature)was obtained by analyzing the data using MicroCal DSC analysis software.

TABLE 15B T-max of the T-max of the Construct Fab fragment part PE24part Humanized SS1 Fab long 73.6 41.2 linker-LRO10R, LRO10R- 456AHumanized SS1 Fab long 73.7 43.2 linker-LRO10R, LRO10R

As shown in FIGS. 12-16 and Table 15B, all chimeric Fab-PE moleculeswith the elongated linker provided a decreased tenedency towardaggregation and a higher aggregation temperature as compared to chimericFab-PE molecules with a short linker that was not elongated.

Example 10

This example demonstrates the ability of deimmunized PE fusion proteinswith an extended linker to kill tumor cells and inhibit cellular proteinsynthesis. This example also demonstrates the antigenicity of thedeimmunized PE fusion proteins with an extended linker.

The potency of different Fab-PE24 variants with respect to the abilityto inhibit protein synthesis was compared. A clone of the pancreaticcancer cell line ASPC-1 (ASPC-1 Luc) that was stably transfected withluciferase was used. The luciferase protein in these cells undergoes ahigh turnover so that the measurable activity drops markedly within 24hours of inhibition of its resynthesis. Briefly, cells were seeded on 96well plates. After overnight culture, different concentrations of thecFP variants were added to the medium, and cells were incubated foranother 72 hours. At the end of the incubation period, the cells werelysed, and luciferase activity was determined with the STEADY-GLOW assayaccording to the manufacturer's instructions. In a typical experiment,an 80-90% reduction of luciferase activity was observed in lysates fromtreated cells as compared to lysates from untreated control cells. EC50values, i.e., the concentrations that achieve half maximal effects, weredetermined based on a free four parameter fit. Table 16 compares therelative potencies in a representative experiment by setting the IC50 ofhuFabLRO10R to 1.

Untargeted PE24 (variant LR8M) only inhibited at concentrations >3.5μg/ml, while the previously described LRO10 variant fused to a humanizedSS1 Fab fragment was ˜500 fold more potent on a molar basis. The LRO10Rbackmutation increased potency 10 to 20 fold (EC50 values 1-2 ng/mlversus 18 ng/ml). The 456A mutation had no adverse effect on potency,while the 551A mutation did reduce activity to the level of LRO10 (EC5010-23 ng/ml). The new extended linker also had no negative impact on thecellular potency of the molecules in a comparison of any of the threepairs of constructs with the extended linker versus those with thepreviously described shorter linker.

Using cell viability assessed by CELLTITERGLOW assays, the cytotoxicpotency of different Fab-PE24 variants using the same humanized SS1targeting variant on two different tumor cell lines (A431 H9 and H596)was compared. Briefly, cells were seeded on 96 well plates. Afterovernight culture, different concentrations of the cFPs were added tothe medium, and cells were incubated for 72 hours. At the end of theincubation period, cell viability was determined using a CELLTITERGLOWassay. Table 16 compares the relative potencies by setting the IC50 ofhuFabLRO10R to 1.

TABLE 16 Viability A431 H9 H596 AsPC-1 assay results Luc Luciferaseassay results cFP variant Fold potency reduction relative to huFabLRO10RhuFabLRO10 1.8 2.5 77.5 LR8M 400 450 9452 huFabLRO10R 1 1 1 huFabLRO10R-1.2 1 2.3 long linker huFabLRO10R- 0.8 1 2.1 456A huFabLRO10R- 0.9 1.2 3456A- longlinker huFabLRO10R- 2.1 3.3 22.5 456A-551A huFabLRO10R- 2.14.5 9.8 456A-551A- longlinker

Results similar to those in Table 16 were also obtained using a cellviability assay with an 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT)-based read-out in different cell lines (Table17).

TABLE 17 IC 50s in ng/ml (did not correct for MW differences) Hu-1 Hu2Hu3 HAY AGS L55 SS1P 2 0.3 2.9 1.0 0.3 3.0 SS1-LR-GGS 0.25 0.01 0.7 — —1.0 SS1-LR-GGS-010R — 0.01 — 0.3 1.0 2.0 Fab-LR-GGS-010R Short 0.25 0.080.4 2.5 0.6 3.5 Fab-LR-GGS-010R 456A 0.25 0.05 0.6 2.5 0.6 3.0 ShortFab-LR-GGS-010R long 0.35 0.04 1.8 2.5 0.9 5.0 Fab-LR-GGS-010R 456A 0.250.04 2.0 1.4 1.1 5.0 long Fab-LR-GGS-010R 456A 0.7 0.18 4.2 4.0 6.0 20.0551A Short Fab-LR-GGS-010R 456A Also Also Also Also Also Also 551A longlow low low low low low

Example 11

This example demonstrates the removal of B-cell epitopes by 456Amuations in PE.

A phagemid containing the scFv 9H3 in the vector pCANTAB as described byLiu et al., PNAS, 109: 11782-11787 (2012) was prepared by standardmethods as described in Sambrook, J. et al., Molecular cloning: Alaboratory manual; Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989. The molecular biological reagents were usedaccording to the manufacturer's instructions. The phagemid wastransformed into electrocompetent TG1 cells. From single colonies,precultures were grown overnight and used to inoculate 4 ml cultures of2×YT/1% glucose supplemented with the appropriate selective antibioticampicillin. At an OD₆₀₀ of 0.3-0.8, 10¹⁰ cfu VCSM 13 helper phage andIPTG was added to a final concentration of 1 mM. After incubation for 15minutes at 37° C., the culture was grown at 37° C. overnight. Two hoursafter helper phage addition, kanamycin was added to final concentrationof 30 μg/ml. The phage-containing supernatant was harvested from theovernight culture by centrifugation, and the scFv displaying phage wereprecipitated from the supernatant by addition of ⅕ v/v 20% PEG-6000/2.5M NaCl and isolated by centrifugation. The phage pellet was dissolved in0.5 ml PBS/1% BSA, and the phage solution was cleared by centrifugationand sterile filtration. The titer of the phage solution was determinedby standard methods as described in Barbas, C. et al., Phage Display—Alaboratory manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 2001.

96 well MaxiSorp microtiter plates were coated with 100 μg/ml Fab-PE24variants. After blocking with PBS buffer supplemented with 2% BSA, 10⁷and 10⁸ scFv-displaying phage were captured on the plate for one hour.As specific negative controls, scFv-displaying phages were pre-incubatedwith 50 μg/m of the Fab-PE24 variants for 1 hour at room temperature(RT) before capturing them on the plate. Plates were washed with PBSTbuffer (PBS+0.5% Tween 20). Binding of scFv-displaying phage wasdetected with POD conjugated anti-m13 antibody (Progen). After a finalwash, the plates were incubated with POD substrate. Absorbance wasmeasured at 405 nm. Results are shown in FIG. 17, where it can be seenthat the B-cell epitope contained in the phagemid containing the scFv9H3 was not removed in LRO10R (with 458R backmutation) while it waseliminated by both mutations 458A and 456A with a slightly strongereffect for the 456A mutation.

Antigenicity testing was done as described in Weldon et al., Mol. CancerTher., 12: 48-57 (2013). As shown in FIGS. 18A and 18B, compared to SS1P(T1), the de-immunized variant LRO10R (T2) had a 100-10,000 fold reducedreactivity with most sera from patients that have been previouslyexposed to a PE-based therapeutic fusion protein both with the murinedsFv fusion format and the humanized Fab fusion format. Also theintroduction of the 456A mutation (T3 and T5) or the new extended linker(T5 and T6) showed a 100-10,000 fold reduced reactivity with most serafrom patients that have been previously exposed to a PE-basedtherapeutic fusion protein.

Example 12

This example demonstrates the construction, expression and purificationof deimmunized PE variants and Fab fusions thereof as well as dsFvfusions for comparison.

Example 12.1 Cloning of Anti-Mesothelin Deimmunized PE ChimericMolecules cFP-0077 (SEQ ID NOs: 41 and 42) and cFP-0078 (SEQ ID NOs: 43and 44)

For the expression of cFP-0077 and cFP-0078, the E. coli host/vectorsystem, which enables an antibiotic-free plasmid selection bycomplementation of an E. coli auxotrophy (PyrF), was employed (EuropeanPatent Application Publication 0 972 838 and U.S. Pat. No. 6,291,245).Standard methods were used to manipulate DNA as described in Sambrook,J. et al., supra. The molecular biological reagents were used accordingto the manufacturer's instructions. Desired gene segments were made bycommercial gene synthesis. The synthesized gene fragments were clonedinto a specified expression vector. The DNA sequences of the subclonedgene fragments were confirmed by DNA sequencing.

The expression plasmids for the production of the light chain and theheavy chain, respectively, were prepared as follows: The light chain(LC) plasmids 15478 (based on the amino acid sequences of cFP-0077 andencoding SEQ ID NO: 41) and 15479 (based on the amino acid sequences ofcFP-0078 and encoding SEQ ID NO: 43) are expression plasmids for theexpression of an antibody light chain fragment in E. coli. They weregenerated by ligating the antibody VL domain fragment into the vectorusing the XhoI/BsiWI restriction sites for 15478, and XhoI/BlpI for15479.

The light chain E. coli expression plasmids comprise the followingelements:

-   -   the origin of replication from the vector pBR322 for replication        in E. coli (corresponding to positions 2517-3160 according to        Sutcliffe et al., Quant. Biol., 43: 77-90 (1979),    -   the URA3 gene of Saccharomyces cerevisiae coding for orotidine        5′-phosphate decarboxylase (Rose et al., Gene, 29:        113-124 (1984) which allows plasmid selection by complementation        of E. coli pyrF mutant strains (uracil auxotrophy),    -   the antibody light chain expression cassette comprising:    -   the T5 hybrid promoter (T5-PN25/03/04 hybrid promoter according        to Bujard et al., Methods. Enzymol., 155: 416-433 (1987) and        Stueber, et al., Immunol. Methods IV, 121-152 (1990) including a        synthetic ribosomal binding site according to Stueber et al.        (supra),    -   the antibody light chain variable domain comprising the CDRs of        SS1, and    -   the human Ck domain for 15478 but not for 15479,    -   two bacteriophage-derived transcription terminators, the λ-T0        terminator (Schwarz et al., Nature, 272: 410-414 (1978) and the        fd-terminator (Beck et al., Gene, 1-3: 35-58 (1981), and    -   the lad repressor gene from E. coli (Farabaugh, Nature, 274:        765-769 (1978).

The heavy chain (HC) plasmids 15476 (based on the amino acid sequencesof cFP-0077 and encoding SEQ ID NO: 42) and 15477 (based on the aminoacid sequences of cFP-0078 and encoding SEQ ID NO: 44) are expressionplasmids for the expression of a fusion protein including an antibodyheavy chain fragment, a linker containing a furin cleavage site, and amutant of domain III of Pseudomonas aeruginosa Exotoxin A (PE), in E.coli. It was generated by ligation of the VH domain fragment into thevector using the XhoI/BsrGI restriction sites for 15476, andXhoI/HindIII restriction sites for 15477.

The cytolytic fusion protein heavy chain E. coli expression plasmidscomprise the following elements:

-   -   the origin of replication from the vector pBR322 for replication        in E. coli (corresponding to positions 2517-3160 according to        Sutcliffe et al., Quant. Biol., 43: 77-90 1979),    -   the URA3 gene of Saccharomyces cerevisiae coding for orotidine        5′-phosphate decarboxylase (Rose et al., Gene, 29:        113-124 (1984) which allows plasmid selection by complementation        of E. coli pyrF mutant strains (uracil auxotrophy),    -   the heavy chain-PE domain III fusion protein expression cassette        comprising    -   the T5 hybrid promoter (T5-PN25/03/04 hybrid promoter according        to Bujard et al., Methods. Enzymol., 155: 416-433 (1987) and        Stueber et al., Immunol. Methods IV, (1990) 121-152) including a        synthetic ribosomal binding site according to Stueber et al.        (supra),    -   the antibody heavy chain variable domain comprising the CDRs of        SS1,    -   the human CH1 domain for 15476 but not for 15477,    -   the linker comprising a furin cleavage site    -   the mutant variant of Pseudomonas aeruginosa Exotoxin A domain        III    -   two bacteriophage-derived transcription terminators, the λ-T0        terminator (Schwarz et al., Nature, 272: 410-414 (1978) and the        fd-terminator (Beck et al., Gene, 1-3: 35-58 (1981), and    -   the lad repressor gene from E. coli (Farabaugh, Nature, 274:        765-769 (1978).

Example 12.2 Expression of Anti-Mesothelin Deimmunized PE ChimericMolecules cFP-0077 and cFP-0078 in E. coli

All polypeptide chains were expressed separately in the E. coli strainCSPZ-6. The E. coli K12 strain CSPZ-6 (thi-1, ApyrF) was transformed byelectroporation with the expression plasmids 15476, 15477, 15478 and15479, respectively, resulting in the 4 strains cFP-0019 (based onplasmid 15476), cFP-0020 (based on plasmid 15477), cFP-0040 (based onplasmid 15478) and cFP-0041 (based on plasmid 15479). For each of thefour strains, the transformed E. coli cells were first grown at 37° C.on agar plates. A colony picked from this plate was transferred to a 3mL roller culture and grown at 37° C. to an optical density of 1-2(measured at 578 nm). Then 1000 μl of culture was mixed with 1000 μlsterile 86%-glycerol and immediately frozen at −80° C. for long timestorage. The correct product expression of this clone was first verifiedin small scale shake flask experiments and analyzed with SDS-Page priorto the transfer to the 10L fermenter.

Pre Cultivation:

For pre-fermentation, a chemically defined medium (CD-PCMv2.20) wasused: NH₄Cl 1.0 g/l, K₂HPO₄*3H₂O 18.3 g/l, citrate 1.6 g/l, Glycine 0.78g/l, L-Alanine 0.29 g/l, L-Arginine 0.41 g/l, L-Asparagine*H₂O 0.37 g/l,L-Aspartate 0.05 g/l, L-Cysteine*HCl*H₂O 0.05 g/l, L-Histidine 0.05 g/l,L-Isoleucine 0.31 g/l, L-Leucine 0.38 g/l, L-Lysine*HCl 0.40 g/l,L-Methionine 0.27 g/l, L-Phenylalanine 0.43 g/l, L-Proline 0.36 g/l,L-Serine 0.15 g/l, L-Threonine 0.40 g/l, L-Tryptophan 0.07 g/l, L-Valine0.33 g/l, L-Tyrosine 0.51 g/l, L-Glutamine 0.12 g/l, Na-L-Glutamate*H₂O0.82 g/l, Glucose*H₂O 6.0 g/l, trace elements solution 0.5 ml/l,MgSO₄*7H₂O 0.86 g/l, and Thiamin*HCl 17.5 mg/l. The trace elementssolution contained FeSO₄*7H₂O 10.0 g/l, ZnSO₄*7H₂O 2.25 g/l, MnSO₄*H₂O2.13 g/l, H₃BO₃ 0.50 g/l, (NH₄)6Mo₇O₂₄*4H₂O 0.3 g/l, CoCl₂*6H₂O 0.42g/l, and CuSO₄*5H₂O 1.0 g/l dissolved in 0.5M HCl.

For pre-fermentation, 220 ml of CD-PCMv2.20 medium in a 1000 mlErlenmeyer-flask with four baffles was inoculated with 1.0 ml out of aresearch seed bank ampoule. The cultivation was performed on a rotaryshaker for 8 hours at 32° C. and 170 rpm until an optical density (578nm) of 2.9 was obtained. 100 ml of the pre cultivation was used toinoculate the batch medium of the 10L bioreactor.

Fermentation of cFP-0019 and cFP-0020:

For fermentation in a 101 Biostat C, DCU3 fermenter (Sartorius,Melsungen, Germany) the following chemically defined batch medium wasused: KH₂PO₄ 1.59 g/l, (NH₄)₂HPO₄ 7.45 g/l, K₂HPO₄*3H₂O 13.32 g/l,citrate 2.07 g/l, L-methionine 1.22 g/l, NaHCO₃ 0.82 g/l, trace elementssolution 7.3 ml/l, MgSO₄*7 H₂O 0.99 g/l, thiamine*HCl 20.9 mg/l,glucose*H₂O 29.3 g/l, biotin 0.2 mg/l, and 1.2 ml/l Synperonic 10% antifoam agent. The trace elements solution contained FeSO₄*7H₂O 10 g/l,ZnSO₄*7H₂O 2.25 g/l, MnSO₄*H₂O 2.13 g/l, CuSO₄*5H₂O 1.0 g/l, CoCl₂*6H₂O0.42 g/l, (NH₄)6Mo₇O₂₄*4H₂O 0.3 g/l, and H₃BO₃ 0.50 g/l solubilized in0.5M HCl solution.

The feed 1 solution contained 700 g/l glucose*H₂O, 7.4 g/l MgSO₄*7 H₂Oand 0.1 g/l FeSO₄*7H₂O. Feed 2 comprises KH₂PO₄ 52.7 g/l, K₂HPO₄*3H₂O139.9 g/l and (NH₄)₂HPO₄ 66.0 g/l. All components were dissolved indeionized water. The alkaline solution for pH regulation was an aqueous12.5% (w/v) NH₃ solution supplemented with 11.25 g/l L-methionine.

Starting with 4.2 l sterile batch medium plus 100 ml inoculum from thepre-cultivation, the batch fermentation was performed at 31° C., pH6.9±0.2, 800 mbar back pressure and an initial aeration rate of 10l/min. The relative value of dissolved oxygen (pO²) was kept at 50%throughout the fermentation by increasing the stirrer speed up to 1500rpm. After the initially supplemented glucose was depleted, indicated bya steep increase in dissolved oxygen values, the temperature was shiftedto 25° C., and 15 minutes later the fermentation entered the fed-batchmode with the start of both feeds (60 and 14 g/h respectively). The rateof feed of 2 was kept constant, while the rate of feed 1 was increasedstepwise with a predefined feeding profile from 60 to finally 160 g/hwithin 7 hours. When carbon dioxide off gas concentration leveled above2%, the aeration rate was constantly increased from 10 to 20 l/minwithin 5 hours. The expression of recombinant protein was induced by theaddition of 2.4 g IPTG at an optical density of approx. 120. The targetprotein was expressed as inclusion bodies within the cytoplasm.

After 24 hours of cultivation, an optical density of 209 was achieved,and the whole broth was cooled down to 4-8° C. The bacteria wereharvested via centrifugation with a flow-through centrifuge (13,000 rpm,13 l/h) and the obtained biomass was stored at −20° C. until furtherprocessing (cell disruption).

Fermentation of cFP-0040 and cFP-0041:

For fermentation in a 10 l Biostat C, DCU3 fermenter (Sartorius,Melsungen, Germany) the following complex batch medium was used:Bacto-Trypton 20 g/l, yeast extract 15 g/l, KH₂PO₄ 1.5 g/l, K2HPO4*3H2O6.6 g/l, NaCl 1.0 g/l, MgSO₄*7 H₂O 0.74 g/l, glucose*H₂O 3.0 g/l, and0.2 ml/l Synperonic 10% anti foam agent.

The feed 1 solution contained Bacto-Trypton 250 g/l and yeast extract175 g/l. pH was controlled using a 75% glucose*H₂O. All components weredissolved in deionized water.

Starting with 7.6 l sterile batch medium plus 100 ml inoculum from thepre cultivation, the batch fermentation was performed at 37° C., pH7.0±0.3, 500 mbar back pressure and an initial aeration rate of 10l/min. The relative value of dissolved oxygen (pO²) was kept at 50%throughout the fermentation by increasing the stirrer speed up to 1500rpm. The feeding of feed 1 was started after the culture reached anoptical density of 15. When carbon dioxide off gas concentration leveledabove 2%, the aeration rate was constantly increased from 10 to 20 l/minwithin 5 hours. The expression of recombinant protein was induced by theaddition of 2.4 g IPTG at an optical density of approx. 20. The targetprotein was expressed insoluble to inclusion bodies within thecytoplasm.

After 13 hours of cultivation, an optical density of 100-115 wasachieved and the whole broth was cooled down to 4-8° C. The bacteriawere harvested via centrifugation with a flow-through centrifuge (13,000rpm, 13 l/h) and the obtained biomass was stored at −20° C. untilfurther processing (cell disruption and inclusion body preparation).

Analysis of Product Formation (for cFP-0019, -0020-0040 and -0041):

Samples were drawn from the fermenter, one prior to induction and theothers at dedicated time points after induction of protein expression,were analyzed with SDS-polyacrylamide gel electrophoresis. From everysample, the same amount of cells (OD_(Target)=10) were suspended in 5 mLPBS buffer and disrupted via sonication on ice. Then 100 μL of eachsuspension were centrifuged (15,000 rpm, 5 minutes), and eachsupernatant was withdrawn and transferred to a separate vial. This wasto discriminate between soluble and insoluble expressed target protein.To each supernatant (=soluble protein fraction) 100 μL and to eachpellet (=insoluble protein fraction) 200 μL of SDS sample buffer(Laemmli, Nature, 227: 680-685 (1970) were added. Samples were heatedfor 15 minutes at 95° C. under intense mixing to solubilize and reduceall proteins in the samples. After cooling to room temperature, 5 μL ofeach sample were transferred to a 4-20% TGX Criterion Stain Freepolyacrylamide gel (Bio-Rad). Additionally, 5 μl molecular weightstandard (Precision Plus Protein Standard, Bio-Rad) were applied.

The electrophoresis was run for 60 minutes at 200 V and thereafter thegel was transferred the GELDOC EZ Imager (Bio-Rad) and processed for 5minutes with UV radiation. Gel images were analyzed using IMAGE LABanalysis software (Bio-Rad). Relative quantification of proteinexpression was done by comparing the volume of the product bands to thevolume of the 25 kDa band of the molecular weight standard.

Inclusion Body Preparation:

The inclusion body preparations (IBP) of the 10L fermentations werestarted directly after the harvest of the bacteria with there-suspension of the harvested bacteria cells in buffer 1 (12.1 g/lTris, 0.246 g/l MgSO4*7H2O, 12 ml/l 25%-HCl). The buffer volume wascalculated in dependence of the dry matter content of the biomass.Lysozyme (100 kU/mg, 0.12 mg/g DCW) and a small amount of benzonase (5U/g DCW) were added. Then the suspension was homogenized at 900 bar (APVRannie 5, 1 pass) to disrupt the bacteria cells followed by the additionof further benzonase (30 U/gDCW) and an incubation for 30 minutes at30-37° C. Then the first wash buffer (60 g/l Brij, 87.6 g/l NaCl, 22.5g/l EDTA, 6 ml/l 10 N NaOH) was added, and again the suspension wasincubated for 30 minutes. The following centrifugation step (BP12,Sorvall) led to the inclusion body slurry, which was re-suspended in thesecond wash buffer (12.1 g/l Tris, 7.4 g/l EDTA, 11 ml/l 25%-HCl) andincubated for 20 minutes. A further separation step harvested theinclusion bodies, which were stored frozen at −20° C. or immediatelysolubilized for refolding and purification.

Example 12.3 Refolding and Purification of Anti-Mesothelin DeimmunizedPE Chimeric Molecules cFP-0077 and cFP-0078

cFP-0077 and cFP-0078 were both obtained by refolding and purification.

Renaturation and Purification of Fab-PE24 (cFP-0077):

Inclusion bodies of HC-PE24 and LC were solubilized separately in 8 MGuanidinium-Hydrochloride, 100 mM Tris/HCl, 1 mM EDTA, pH 8.0+100 mMDithiothreitol (DTT) overnight at RT (1 g IB in 5 mL). Solubilizateswere adjusted to pH 3 and centrifuged, and the pellet was discarded.After extensive dialysis against 8 M Guanidinium-HCl, 10 mM EDTA, pH 3.0to remove DTT, the total protein concentration was determined using theBiuret method. The purity of HC and LC content was estimated viaSDS-PAGE.

Solubilizates were diluted at a 1:1 molar ratio in renaturation buffercontaining 0.5 M arginine, 2 mM EDTA, pH 10+0.9 mM GSH/GSSG,respectively, at 2-10° C. The target protein concentration was increasedin two steps to 0.22 g/L, with an incubation time of 4 h between the twodoses. Afterwards, the renaturation solution was kept at 2-10° C.overnight.

The renaturate was diluted with H₂O to <3 mS/cm and pumped onto an anionexchange column (AIEX) equilibrated in 20 mM Tris/HCl, pH 7.4. Afterwashing the column with equilibration buffer, the protein was elutedwith a gradient up to 20 mM Tris/HCl, 400 mM NaCl, pH 7.4. Peakfractions containing Fab-PE24 were pooled, concentrated and applied ontoa preparative Size Exclusion Column (SEC) in 20 mM Tris, 150 mM NaCl, pH7.4 to remove aggregates, fragments and E. coli proteins. The finalprotein pool was adjusted to the required protein concentration andanalyzed via SDS-PAGE, analytical SEC and UV₂₈₀, and identity wasconfirmed by mass spectrometry.

Renaturation and Purification of dsFv-PE24 (cFP-0078):

Inclusion bodies of HC-PE24 and LC were solubilized separately 8 MGuanidinium-Hydrochloride, 100 mM Tris/HCl, 1 mM EDTA, pH 8.5+100 mM DTTovernight at RT (1 g IB in 5 mL). Solubilisates were adjusted to pH 3and centrifuged, and the pellet was discarded. After extensive dialysisagainst 7.2 M Guanidinium-HCl, 10 mM EDTA, pH 3.0 to remove DTT, thetotal protein concentration was determined using the Biuret method, andthe purity of HC and LC content was estimated via SDS-PAGE.

Solubilizates were diluted 1:100 in renaturation buffer containing 0.5 Marginine, 2 mM EDTA, pH 10+0.9 mM GSH/GSSG, respectively, at a 1:1 molarratio and kept overnight at 2-10° C.

The renaturate was diluted with H₂O to <3 mS/cm and pumped onto an anionexchange column (AIEX) equilibrated in 20 mM Tris/HCl, 1 mM EDTA, pH7.4. After washing the column with equilibration buffer, the protein waseluted with a gradient up to 20 mM Tris/HCl, 1 mM EDTA, 400 mM NaCl, pH7.4. Peak fractions containing dsFv-PE24 were pooled, concentrated andapplied onto a preparative Size Exclusion Column (SEC) in 20 mMTris/HCl, 150 mM NaCl, pH 7.4 to remove aggregates, fragments and E.coli proteins. The final protein pool was adjusted to the requiredprotein concentration and analyzed via CE-SDS, analytical SEC und UV₂₈₀.Identity was confirmed by mass spectrometry.

Example 12.4 Protein Analysis of cFP-0077 and cFP-0078

Samples were analyzed by OD 280 nm using a UV spectrophotometer todetermine the protein concentration in solution. The extinctioncoefficient required for this was calculated from the amino acidsequence according to Pace et al., Protein Science, 4: 2411-2423 (1995).Size-exclusion chromatography (SE-HPLC) was performed on TSK-Gel300SWXLor Superdex 200 columns with a 0.2 M potassium phosphate buffer,comprising 0.25 M KCl, pH 7.0 as mobile phase in order to determine thecontent of monomeric, aggregated and degraded species in the samples.Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis(reducing and non-reducing) was performed to analyze the purity of thecomplex preparations with regard to product-related degradation productsand unrelated impurities (for details, see below). Electrosprayionisation mass spectrometry (ESI-MS) was performed with reduced (TCEP)samples to confirm the correct mass/identity of each chain and detectchemical modifications. ESI-MS of the non-reduced samples was carriedout to analyze the nature and quality of the fully assembled protein anddetect potential product-related side products.

Method for SDS-PAGE and Coomassie Staining Device: Invitrogen XCELL SURELOCK Mini-Cell Gel: 4-20% Tris-Glycine Gel, Invitrogen EC6025BOX Buffer:Tris-Glycine SDS Running Buffer (10×), Invitrogen LC2675-5

Sample buffer: Tris-Glycine SDS Sample Buffer (2×), Invitrogen LC2676Reducing buffer: NuPAGE Sample Reducing Agent (10×), Invitrogen NP0004

Molecular Weight Marker: Mark 12, MW Standard, Invitrogen LC5677

The sample was adjusted to a protein concentration of 1 mg/ml withbuffer. For sample reduction, the following procedure was carried out: Areduction buffer including 4 ml Sample buffer (2×) and 1 ml reducingbuffer (10×) was prepared. The sample was diluted 1:1 with reductionbuffer and incubated for 5 minutes at 70° C.

The gel electrophoresis was carried out at 125 V for 90 minutes. Thegels were stained with SIMPLY BLUE Safe Stain (Invitrogen, Cat. No.LC6065).

Example 12.5 Comparison of Fab Vs dsFv Fusion Protein-CytotoxicProperties

The cytotoxic potency of a dsFv-PE24 fusion protein format and aFab-PE24 format was compared using the same humanized SS1 targetingmoiety on three different tumor cell lines (MKN45, H596, and A431H9).Briefly, cells were seeded on 96 well plates. After overnight culture,different concentrations of the cFPs were added to the medium and cellswere incubated for 72 h. At the end of incubation period, cell viabilitywas determined using a CELLTITERGLOW assay.

In each case, the dose-response curves and IC50 values for inhibition ofcell viability were similar for the dsFv-PE24 and the Fab-PE24 format.The molar ratio of the IC50 values is listed in Table 18. Depending onthe cell line tested, the targeted PE24 fusion proteins were severalhundred to one thousand fold more potent than untargeted PE24.

TABLE 18 Ratio of the molar IC50 values Cell line dsFv-PE24/Fab-PE24A431H9 0.37 MKN45 2.3 H596 0.38

Example 12.6 Comparison of Fab Versus dsFv Fusion Protein-Serum Kinetics

The serum kinetics of the following three Pseudomonas exotoxin A(PE)-based anti-mesothelin immunotoxins in SCID beige mice was compared:SS1dsFv-PE38 (MW: 62.5 kDa), SS1dsFv-PE24 (MW: 49.9 kDa) and SS1Fab-PE24(MW: 72.2 kDa). The murine SS1 antibody moiety was used for allconstructs. The kinetics were compared at doses equimolar to 0.2 mg/kg(3.2 nmol/kg) SS1P) corresponding to 0.231 mg/kg SS1Fab-PE24, and 0.160mg/kg for SS1dsFv-PE24.

The constructs were administered as a single intravenous dose, and theirserum levels were analyzed at 0.08, 0.5, 1, 1.5, 2, 3, 4, 5, and 7 hoursafter dosing. Nine mice per group were administered the studymedication, and blood was collected in a staggered manner at three timesper animal by retro-orbital puncture, resulting in nine time points withn=3 samples. Serum samples were analyzed by an ELISA that detects onlyintact test compounds having the antibody fragment plus the PE part. Inthis assay format, the cFP is captured on a MSLN-coated surface and thePE moiety is detected with a rabbit polyclonal anti-PE antibody andbiotinylated anti-rabbit secondary antibody.

For each time point, average serum values from three animals wereplotted for the treatment groups together with historical data fromCD2F1 mice. The SS1dsFv-PE24 construct was very rapidly cleared with aserum half-life of only ˜12 minutes. In agreement with the historicaldata, clearance of SS1P was much slower, resulting in a serum half lifeof between 35 and 44 minutes (values derived from measured andhistorical data, respectively). The pharmacokinetic properties of theSS1Fab-PE24 construct were very similar to SS1dsFv-PE38 with a serumhalf life of 43 minutes and similar values were also obtained forclearance rate, volume of distribution, c_(max) and area under the curve(Table 19 and FIG. 19).

TABLE 19 Comparison Fab versus dsFv fusion protein- Serum kineticsParameter Unit SS1P SS1-Fab-PE24 SS1-dsFv-PE24 C_(L) mL/(min · kg) 2.241.53 7.06 V_(C) mL/kg 84.3 75.4 146 V_(SS) mL/kg 92 80.5 103 T_(1/2) h0.574 0.716 0.201 C_(max) ng/mL 1990 2730 837 AUC ng · h/mL 1490 2520378 (0-inf)

Example 13

This example demonstrates the humanization of the anti-mesothelinantibody SS1.

Example 13.1 Design of Humanized Heavy and Light Chain Variable Regions

The structures of the VH and the VL domain of the SS1 antibody weremodeled in silico, and the model was compared to a structural databaseof human VH and VL domains. A panel of the most structurally similar Vdomains were chosen for grafting the CDRs of SS1 onto the human VH andVL domains. In addition, similarities in the primary sequence were takeninto account to narrow down the choice of the human V domains byaligning the primary sequence of the VH and VL domain of SS1 to thehuman V domain repertoire. Backmutations within the human frameworkregions to mouse parent residues were introduced in some humanizationvariants. Similarly, mutations in the CDRs were introduced in somevariants, where appropriate, to potentially increase the affinity to theantigen or to maintain the CDR tertiary structure.

The designed V domain variants were cloned into heavy chain and lightchain vectors 6318 and 6319 via unique cloning sites to generate humanIgG light and heavy chains. The heavy and light chain vectors wereco-transfected into HEK293 suspension cells in microtiter culture platesin a matrix manner to obtain cell cultures expressing full size IgGhaving all possible light/heavy chain combinations. After 5 days,cultivation at 37° C., the supernatants were harvested and purified byProtein A affinity chromatography in the microtiter scale.

Example 13.2 Cloning of Humanized Heavy and Light Chain Variable Regions

Standard methods were used to manipulate DNA as described in Sambrook,J. et al., supra. The molecular biological reagents were used accordingto the manufacturer's instructions. Desired gene segments were preparedby commercial gene synthesis. The synthesized gene fragments were clonedinto a specified expression vector. The DNA sequence of the subclonedgene fragments were confirmed by DNA sequencing.

Expression Vector for the Antibody Heavy Chains:

The gene segments of the designed humanized antibody heavy chainvariable domains were cloned into the specified expression vector viathe unique restriction sites HindIII and XhoI. The expression vector wasdesigned to express the antibody heavy chain variable domain in fusionwith the human antibody domains CH1, hinge, CH2 and CH3 in HEK293 cells,resulting in a conventional antibody heavy chain. All domains wereseparated by introns. Besides the expression cassette for the antibodyheavy chain, the vector contained:

-   -   an origin of replication from the vector pUC18 which allows        replication of this plasmid in E. coli,    -   a β-lactamase gene which confers ampicillin resistance in E.        coli,    -   an SV40 promotor and origin for expression of the DHFR selection        marker,    -   Murine dihydrofolate reductase (DHFR) as selection marker for        antibody expression, and    -   SV40′ early polyadenylation (“early poly A”) signal sequence.

The transcription unit of the antibody heavy chain is composed of thefollowing elements:

-   -   the immediate early enhancer and promoter from the human        cytomegalovirus,    -   a 5′-untranslated region of a human antibody germline gene,    -   a murine immunoglobulin heavy chain signal sequence including a        signal sequence intron (signal sequence 1, intron, signal        sequence 2 [L1-intron-L2]),    -   the VH sequence followed by an intron,    -   human CH1, hinge, CH2, CH3 domains separated by introns,    -   the bovine growth hormone (bGH) polyadenylation (“poly A”)        signal sequence, and    -   the human gastrin transcription terminator (HGT).

Expression Vector Fort he Antibody Light Chains:

The gene segments of the designed humanized antibody light chainvariable domains were cloned into a specified expression vector via theunique restriction sites BsmI and Celt The expression vector wasdesigned to place the antibody light chain variable domain in fusionwith the human antibody domain Ck in HEK293 cells. Both domains wereseparated by an intron. Besides the expression cassette for the antibodylight chain, the vector contains:

-   -   an origin of replication from the vector pUC18 which allows        replication of this plasmid in E. coli, and    -   a β-lactamase gene which confers ampicillin resistance in E.        coli.

The transcription unit of the antibody light chain is composed of thefollowing elements:

-   -   the immediate early enhancer and promoter from the human        cytomegalovirus,    -   a 5′-untranslated region of the human cytomegalovirus,    -   an Intron A sequence of the human cytomegalovirus,    -   a 5′-untranslated region of a human antibody germline gene,    -   a murine immunoglobulin heavy chain signal sequence including a        signal sequence intron (signal sequence 1, intron, signal        sequence 2 [L1-intron-L2]) and the unique restriction site BsmI        at the 3′ end of L2,    -   a VL domain followed by an intron,    -   human Ck domain,    -   the bovine growth hormone (bGH) polyadenylation (“poly A”)        signal sequence, and    -   Human gastrin transcription termination factor (HGT).

Example 13.3 Expression of Humanized Antibodies

Transient transfection of humanized antibody light and heavy chains:Recombinant humanized antibody variants were generated by transienttransfection of HEK293-Freestyle cells (human embryonic kidney cell line293, Invitrogen) grown in suspension. The transfected cells werecultivated in F17 medium (Gibco) or Freestyle 293 medium (Invitrogen),either one supplemented with 6 mM glutamine, either ultra-glutamine(Biowhittake/Lonza) or L-glutamine (Sigma), with 8% CO₂ at 37° C. inshake flasks in the scale of 2 ml medium in 48-deep-well plates to 1 Lmedium in shake flasks. 293-Free transfection reagent (Novagen/Merck)was used in a ratio of reagent (μl) to DNA (μg) of 4:3. Light and heavychains were expressed from two different plasmids using a molar ratio oflight chain to heavy chain encoding plasmid ranging from 1:2 to 2:1,respectively. Humanized antibody containing cell culture supernatantswere harvested at day 6 to 8 after transfection. General informationregarding the recombinant expression of human immunoglobulins in, e.g.,HEK293 cells, is given in: Meissner et al., Biotechnol. Bioeng., 75:197-203 (2001). The supernatants were purified by affinitychromatography on MabSelect Sure (GE, Protein A) equilibrated in PBS.After application of the filtrated supernatants, the antibodies wereeluted with 50 mM sodium acetate, pH 3.2. The pH of the eluates wasadjusted immediately to pH>6 with 2 M Tris/HCl, pH 9.0 followed bydialysis into 20 mM histidine, 140 mM NaCl, pH 6.0. Antibodies wereanalyzed by UV₂₈₀, SDS-PAGE and analytical SEC. The sequence wasconfirmed by mass spectrometry.

Example 13.4 Characterization of Humanized SS1 Variants

ELISA screening of humanized SS1 variants: All SS1 humanization variantsin the IgG format were screened for affinity by ELISA. 384 well MaxiSorpmicrotiter plates were coated with 0.5 μg/ml anti-His antibody(Novagen). After blocking with PBS buffer supplemented with 2% BSA, 0.1%Tween 20, and 0.2 μg/ml human/cynomolgus/murine mesothelin (in house/R&DSystems), all proteins with a His-tag were captured on the plate for onehour. The plates were washed with PB ST Buffer (PBS+0.1% Tween 20) anddilutions of humanized anti-mesothelin antibodies in PBS were incubatedfor 1 hour at room temperature. Binding of antibodies was detected withHRP conjugated anti human Fc antibody (GE Healthcare). After a finalwash, the plates were incubated with HRP substrate. Absorbance wasmeasured at 370 nm on an ENVISION plate reader. EC50 curve fit analysiswas performed using XLfit4 analysis plug-in for EXCEL software (model205).

To normalize the obtained values to the IgG titer, quantitation of humanIgG was performed by sandwich ELISA using streptavidin coated plates.Streptavidin-coated 384 well microtiter plates (Microcoat) wereincubated with a mixture of 0.25 μg/ml biotinylated anti-human IgGantibody (Jackson Imm. Res.), 0.05 μg/ml anti-human IgG-HRP conjugate(Jackson Imm. Res.), and dilutions of the humanized ant-mesothelinantibodies. After 1.5 hours incubation at room temperature (RT), plateswere washed with PBS buffer supplemented with 0.1% Tween 20. HRPsubstrate was added, and the absorbance was measured at 370 nm on anENVISION plate reader. The calculation of data was performed using ahuman IgG reference as a standard (in house) for calibration and XLfit4analysis plug-in for EXCEL software (model 205) for curve fit analysis.

In Tables 20-21 below, the ELISA data of all humanization variants areshown. ELISA/BIACORE binding to huMesothelin (EC50 ng/ml) was measured,and the results are shown in Table 20 (in house) and Table 21 (R&D).Normalized to IgG titer, this primary screen identified the variantVL001/VH001 as one of the combinations with the best EC50 value.

BiaCore Screening of Humanized SS1 Variants:

The best variants containing CDRs most similar to SS1 were chosen forkinetics analysis by surface plasmon resonance (SPR). An SPR based assayhas been used to determine the kinetic parameters of the binding betweenseveral MSLN PE cFP humanization variants and human mesothelin.Therefore, Protein A was immobilized by amine coupling to the surface ofthe CMS biosensor chip. The samples were then captured, and humanmesothelin was injected. The sensor chip surface was regenerated betweeneach analysis cycle. The equilibrium constant K_(D) as well as the rateconstants k_(d) and k_(a) were finally gained by fitting the data to a1:1 langmuir interaction model. About 175 response units (RU) of ProteinA (10 μg/ml) were coupled onto the CMS sensor chip at pH 4.0 by using anamine coupling kit supplied by GE Healthcare (10 minutes activation).The sample and system buffer was HBS-P+(0.01 M HEPES, 0.15 M NaCl,0.005% surfactant P20 sterile-filtered, pH 7.4). The flow celltemperature was set to 25° C., and the sample compartment temperaturewas set to 12° C. The system was primed with running buffer. The proteinA binding sites were saturated with IgGs (about 0.03 μg/mL) to generatesimilar capture levels for each sample, by injecting them for 40 secondsat a flow rate of 10 μl/min. Afterwards, a single 50 nM human mesothelinsolution was injected for 120 seconds at a flow rate of 30 μl/min,followed by a 180 second dissociation phase. Thereby a relative KDdetermination allowed a ranking of different MSLN binders. Each cyclewas regenerated with two injections of glycine-HCl pH 1.5 (30 seconds,30 μl/min).

Table 22 below lists the BiaCore data of 12 selected humanizationvariants. The final humanization variant VL01/VH01 shows one of thelowest Kd values among all humanization variants. Table 22 shows theBiacore results for cFP.15438-15457 and cFP.15438-15459, all comprisinghumanized VH1. cFP.15438, which comprises the amino acid sequence of SEQID NO: 45, displays the highest affinity of the humanized variants ofSS1 antibody. Thus, the VH of cFP.15438 provides advantageous bindingproperties.

The five best variants were cloned into the Fab-Linker-PE formatcontaining the published linker and LRO10 as the PE24 variant,expressed, refolded and purified. Cloning, expression, refolding andpurification were carried out as described below for cFP-0205. The fivehumanization variants in the cytolytic fusion format were analyzed forstability at 37° C. for 7 days by the method of dynamic light scatteringdescribed in Example 9.2, size exclusion chromatography, SDS-PAGE, andmass spectrometry at distinct time points.

Size-exclusion chromatography (SE-HPLC) was performed on TSK-Gel300SWXLor Superdex 200 columns with a 0.2 M potassium phosphate buffercomprising 0.25 M KCl (pH 7.0) as the mobile phase in order to determinethe content of monomeric, aggregated, and degraded species in thesamples. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis(reducing and non-reducing) was performed to analyze the purity of thecomplex preparations with regard to product-related degradation productsand unrelated impurities (for details, see below). Electrosprayionisation mass spectrometry (ESI-MS) was performed with reduced (TCEP)samples to confirm the correct mass/identity of each chain and to detectchemical modifications. ESI-MS of the non-reduced samples was carriedout to analyze the nature and quality of the fully assembled protein andto detect potential product-related side products.

The active concentration of the Fab fragments within the cytolyticfusion format after temperature stress at 37° C. for 7 days wasmonitored by surface plasmon resonance (SPR). Mesothelin was immobilizedonto the surface of a SPR biosensor. By injecting the sample into theflow cells of the SPR spectrometer, it formed a complex with theimmobilized mesothelin, resulting in an increased mass on the sensorchip surface and, therefore, a higher response (as 1 RU is defined as 1pg/mm²). Afterwards, the sensor chip is regenerated by dissolving thesample-mesothelin-complex. The gained responses are then evaluatedrelative to the response displayed by the reference standard (which isassumed to be 100% active). First, around 50 resonance units (RU) ofhuman MSLN (0.75 μg/ml) were coupled on a C1 chip (GE Healthcare) at pH4.5 by using the amine coupling kit of GE Healthcare. The sample andsystem buffer was HBS-P+ (0.01 M HEPES, 0.15 M NaCl, 0.005% surfactantP20 sterile-filtered, pH 7.4). The flow cell temperature was set to 25°C., and the sample compartment temperature was set to 12° C. The systemwas primed with running buffer. Then, a 5 nM solution of the MSLN PE cFPconstruct was injected for 60 seconds at a flow rate of 30 μl/min,followed by a 60 second dissociation phase. Then, the sensor chipsurface was regenerated by two 20 second long injections of theregeneration solution (0.31M KSCN, 1.22M MgCl₂, 0.61M urea, 1.22MGua-HCl, 6.7 mM EDTA) at a flow rate of 30 μl/min, followed by an extrawash step with buffer and a 5 second stabilization period.

Stability data were obtained by incubating the 5 best humanized variantsin the Fab-linker-PE24LRO10 format at 37° C. for 7 days. The results areshown in Table 23. As shown in Table 23, the variant VH01/VL01 was theonly variant that showed no fragmentation after temperature stress. TheVL01 promoted recovery as all VL01 combinations formed only aggregatesafter temperature stress but did not precipitate. Negative results ofaffinity measurements after temperature stress were due to theseaggregates. Accordingly, the humanization variant VH1/VL1 (SEQ ID NO:45)/(SEQ ID NO: 46) was chosen for further development. VH1 (SEQ ID NO:45) provided favorable functionality and binding properties. Thecombination of VH1 (SEQ ID NO: 45)/VL1 (SEQ ID NO: 46) providedfavorable developability.

TABLE 20 z a b c d VL 000/ 001/ 15456 15457 mouse (SEQ ID 002/ 003/ 004/mVL NO: 46) 15458 15459 15460 Past humanized VH (C99Q) IMGT_hVK_1_39IMGT_hVK_3_11 IMGT_hVK_1_39 Hercept. Mouse 0 000/15437 mVH Past 20.1017.80 13.36 20.80 32.72 (C44Q) Humanized 1 001/15438 IMGT_hVH_1_46 32.0420.27 15.57 22.44 28.95 (SEQ ID NO: 45) 2 002/15439 VBase_VH1_1 88.85204.74 32.01 48.39 38.63 3 003/15440 VBase_VH1_1 37.76 100.18 31.5839.86 63.66 4 004/15441 VBase_VH1_1 39.46 46.18 20.21 52.31 112.42 5005/15442 Herceptin 71.84 432.87 39.39 70.55 94.99 6 006/15443IMGT_hVH_5_51 196.59 287.08 41.11 144.51 466.56 7 007/15444IMGT_hVH_5_51 155.21 67.47 137.39 71.03 >500.0 8 008/15445 IMGT_hVH_5_51431.47 44.39 42.37 17.40 143.88 9 009/15446 IMGT_hVH_1_8 118.32 62.2371.58 31.00 230.84 10 010/15447 IGHV4-34-05 41.35 93.85 35.06 33.49135.83 11 011/15448 IMGT_hVH_3_21 44.04 52.11 24.52 41.68 54.65 12012/15449 VBase_VH1_1 30.19 37.51 7.86 47.87 36.40 13 013/15450VBase_VH1_1 70.85 >500.0 58.84 129.80 111.42 14 014/15451 VBase_VH1_1#N/A #N/A #N/A #N/A #N/A M1 MSAb- Morphotek 25.65 19.12 13.24 18.8233.79 1/15452 M2 MSAb- Morphotek 23.42 15.51 33.37 18.46 30.84 2/15453M3 MSAb- Morphotek 34.70 62.58 17.71 15.82 64.02 3/15454 SS1HC_SS1/15436 Pastan 47.15 38.16 26.40 28.85 60.38 e m1 m2 m3 ss1 VL 005/MSAb- MSAb- MSAb- LC_SS1/ 15461 1/15462 2/15463 3/15464 15455 humanizedMorphotek Morphotek Morphotek Pastan VH IMGT_hVK_1_39 PAT PAT PAT PATMouse 0 000/15437 mVH Past 20.15 16.89 26.38 13.89 17.42 (C44Q)Humanized 1 001/15438 IMGT_hVH_1_46 30.31 33.50 21.78 21.23 27.22 (SEQID NO: 45) 2 002/15439 VBase_VH1_1 103.46 181.39 81.92 47.73 271.77 3003/15440 VBase_VH1_1 70.03 26.60 108.18 39.95 230.87 4 004/15441VBase_VH1_1 68.93 73.53 71.82 89.99 270.06 5 005/15442 Herceptin 119.5226.06 326.88 60.33 >500.0 6 006/15443 IMGT_hVH_5_51 163.55 >500.0 83.6178.58 52.29 7 007/15444 IMGT_hVH_5_51 69.42 210.52 89.05 182.10 181.13 8008/15445 IMGT_hVH_5_51 25.90 20.03 16.34 37.09 87.91 9 009/15446IMGT_hVH_1_8 68.65 103.29 97.08 80.73 44.37 10 010/15447 IGHV4-34-0595.84 66.42 65.63 26.84 235.42 11 011/15448 IMGT_hVH_3_21 62.08 15.60124.88 35.08 47.05 12 012/15449 VBase_VH1_1 61.27 30.13 35.53 20.6886.50 13 013/15450 VBase_VH1_1 132.05 16.51 204.88 33.20 >500.0 14014/15451 VBase_VH1_1 #N/A #N/A #N/A #N/A #N/A M1 MSAb- Morphotek 21.7126.82 13.24 17.05 7.37 1/15452 M2 MSAb- Morphotek 30.16 39.31 23.7535.77 30.54 2/15453 M3 MSAb- Morphotek 53.63 117.75 19.86 42.60 45.233/15454 SS1 HC_SS1/15436 Pastan 37.42 29.65 29.79 14.77 41.13

TABLE 21 z a b c VL 000/ 001/ 15456 15457 mouse (SEQ ID 002/ 003/ mVLNO: 46) 15458 15459 Past humanized VH (C99Q) IMGT_hVK_1_39 IMGT_hVK_3_11IMGT_hVK_1_39 Mouse 0 000/15437 mVH Past (C44Q) 24.93 18.25 14.91 16.97Humanized 1 001/15438 (SEQ IMGT_hVH_1_46 22.81 15.55 16.22 21.03 ID NO:45) 2 002/15439 VBase_VH1_1 20.80 30.71 9.67 22.61 3 003/15440VBase_VH1_1 11.17 64.84 21.95 24.52 4 004/15441 VBase_VH1_1 13.30 18.7911.38 20.34 5 005/15442 Herceptin 57.64 75.30 21.79 26.38 6 006/15443IMGT_hVH_5_51 37.98 68.72 17.00 31.68 7 007/15444 IMGT_hVH_5_51 30.9232.33 50.56 47.93 8 008/15445 IMGT_hVH_5_51 27.84 32.16 13.63 17.70 9009/15446 IMGT_hVH_1_8 37.15 20.48 23.00 9.24 10 010/15447 IGHV4-34-0517.19 36.94 18.55 3.79 11 011/15448 IMGT_hVH_3_21 17.85 19.31 12.0926.23 12 012/15449 VBase_VH1_1 26.50 18.36 4.84 25.45 13 013/15450VBase_VH1_1 17.11 48.46 26.21 32.81 14 014/15451 VBase_VH1_1 #N/A #N/A#N/A #N/A M1 MSAb-1/15452 Morphotek 16.76 26.05 10.85 15.40 M2MSAb-2/15453 Morphotek 18.94 13.24 25.61 18.06 M3 MSAb-3/15454 Morphotek43.79 80.00 24.10 20.13 SS1 HC_SS1/15436 Pastan 34.77 37.48 30.64 28.76d e m1 m2 m3 ss1 VL 004/ 005/ MSAb- MSAb- MSAb- LC_SS1/ 15460 154611/15462 2/15463 3/15464 15455 humanized Morph Morph Morph Pastan VHHercept IMGT_hVK_1_39 PAT PAT PAT PAT Mouse 0 000/15437 mVH Past (C44Q)28.24 19.65 13.30 20.08 16.62 21.59 Humanized 1 001/15438 (SEQIMGT_hVH_1_46 14.77 21.75 33.91 21.69 18.48 20.27 ID NO: 45) 2 002/15439VBase_VH1_1 18.10 23.88 26.61 30.97 26.83 23.16 3 003/15440 VBase_VH1_127.66 46.86 11.40 33.78 23.40 43.11 4 004/15441 VBase_VH1_1 20.71 23.0618.22 20.33 19.35 35.09 5 005/15442 Herceptin 22.71 32.75 7.46 78.6041.55 51.40 6 006/15443 IMGT_hVH_5_51 96.41 77.48 59.94 13.32 37.23 9.707 007/15444 IMGT_hVH_5_51 97.81 21.06 35.60 49.19 35.21 26.52 8008/15445 IMGT_hVH_5_51 36.39 14.14 8.96 12.58 15.26 14.19 9 009/15446IMGT_hVH_1_8 99.49 45.65 13.29 16.81 20.11 11.92 10 010/15447IGHV4-34-05 33.67 29.20 15.63 19.91 14.16 58.47 11 011/15448IMGT_hVH_3_21 19.38 25.78 6.71 42.47 25.88 26.04 12 012/15449VBase_VH1_1 32.87 32.83 22.30 16.38 21.03 30.01 13 013/15450 VBase_VH1_117.97 55.13 18.76 40.91 16.60 70.42 14 014/15451 VBase_VH1_1 #N/A #N/A#N/A #N/A #N/A #N/A M1 MSAb-1/15452 Morphotek 27.67 15.26 18.45 10.0714.15 10.22 M2 MSAb-2/15453 Morphotek 18.24 32.68 49.52 33.94 33.7231.20 M3 MSAb-3/15454 Morphotek 57.15 51.29 76.89 21.74 57.13 43.21 SS1HC_SS1/15436 Pastan 50.76 48.23 24.34 31.68 17.66 46.14

TABLE 22 Antibody Antigen Curve Temp (° C.) Fit ka kd KD KD [nM]cFP-0004-0002 Mesothelin Fc = 4-3 25 1:1 Binding 7.88E+05 2.39E−043.04E−10 0.30 cFP-0006-0002 Mesothelin Fc = 4-3 25 1:1 Binding 9.29E+052.62E−04 2.83E−10 0.28 cFP.15438-15457 Mesothelin Fc = 4-3 25 1:1Binding 8.39E+05 2.20E−04 2.63E−10 0.26 cFP.15438-15459 Mesothelin Fc =4-3 25 1:1 Binding 7.13E+05 1.97E−04 2.77E−10 0.28 cFP.15438-15460Mesothelin Fc = 4-3 25 1:1 Binding 4.92E+05 1.63E−04 3.31E−10 0.33cFP.15442-15457 Mesothelin Fc = 4-3 25 1:1 Binding 2.15E+05 0.0013876.45E−09 6.45 cFP.15442-15459 Mesothelin Fc = 4-3 25 1:1 Binding2.25E+05 0.002445 1.09E−08 10.89  cFP.15442-15460 Mesothelin Fc = 4-3 251:1 Binding 1.42E+05 0.002221 1.57E−08 15.70  cFP.15447-15457 MesothelinFc = 4-3 25 1:1 Binding 6.31E+05 9.76E−04 1.55E−09 1.55 cFP.15447-15459Mesothelin Fc = 4-3 25 1:1 Binding 5.14E+05 0.001916 3.73E−09 3.73cFP.15447-15460 Mesothelin Fc = 4-3 25 1:1 Binding 3.55E+05 0.0020695.82E−09 5.82 cFP.15448-15457 Mesothelin Fc = 4-3 25 1:1 Binding1.77E+05 0.001729 9.79E−09 9.79 cFP.15448-15459 Mesothelin Fc = 4-3 251:1 Binding 1.73E+05 0.001909 1.11E−08 11.05  cFP.15448-15460 MesothelinFc = 4-3 25 1:1 Binding 1.12E+05 0.00216 1.93E−08 19.32 

TABLE 23 T_(agggreagetion) Target Stress effect on VL VH [° C.] RecoverySEC SDS-PAGE ESI-MS d ESI-MS d&r binding binding VL01 VH10 40 + HMWFragmentation Fragmentation. Fragmentation. +/− HMW VL03 VH01 ~35 − LMWFragmentation. Fragmentation. Fragmentation. + +/− VL01 VH01 37 + HMWsNo No No + HMW fragmentation fragmentation fragmentation VL03 VH10 37 −HMW +/− Fragmentation. Fragmentation. +/− +/− VL04 VH01 34 − LMW +/− NoNo + + fragmentation fragmentation

Example 14

This example demonstrates the cloning, expression, refolding, andpurification of chimeric deimmunized PE LOR10-456A as a Fab fusion withhumanized anti-mesothelin SS1 (cFP-0205).

The chimeric PE anti-mesothelin molecule cFP-0205 was obtained aftercloning, expression, refolding, and purification. cFP-0205 (alsoreferred to as R205 or RG7787) comprises amino acid sequences SEQ ID NO:39 and 40.

Cloning of cFP-0205:

For the expression of cFP-0205, the E. coli host/vector system whichenables an antibiotic-free plasmid selection by complementation of an E.coli auxotrophy (PyrF) was employed (EP 0 972 838 and U.S. Pat. No.6,291,245). Standard methods were used to manipulate DNA as described inSambrook, J. et al., supra. The molecular biological reagents were usedaccording to the manufacturer's instructions. Desired gene segments weremade by commercial gene synthesis. The synthesized gene fragments werecloned into a specified expression vector. The DNA sequences of thesubcloned gene fragments were confirmed by DNA sequencing.

The expression plasmids for the production of the light chain (LC) andthe heavy chain (HC), respectively, were prepared as follows: The LCPlasmid 15496 is an expression plasmid for the expression of an antibodylight chain in E. coli. It was generated by ligating the antibody VLdomain fragment into the vector using the NdeI/BsiWI restriction sites.

The light chain E. coli expression plasmid comprises the followingelements:

-   -   the origin of replication from the vector pBR322 for replication        in E. coli (corresponding to positions 2517-3160 according to        Sutcliffe et al., Quant. Biol., 43: 77-90 (1979),    -   the URA3 gene of Saccharomyces cerevisiae coding for orotidine        5′-phosphate decarboxylase (Rose et al., Gene, 29: 113-124        (1984), which allows plasmid selection by complementation of E.        coli pyrF mutant strains (uracil auxotrophy),    -   the antibody light chain expression cassette, comprising    -   the T5 hybrid promoter (T5-PN25/03/04 hybrid promoter according        to Bujard et al., Methods. Enzymol., 155: 416-433 (1987) and        Stueber et al., Immunol. Methods IV, 121-152 (1990) including a        synthetic ribosomal binding site according to Stueber et al.        (supra),    -   the antibody light chain variable domain comprising the CDRs of        SS1,    -   the human Ck domain,    -   two bacteriophage-derived transcription terminators, the λ-T0        terminator (Schwarz et al., Nature, 272: 410-414 (1978) and the        fd-terminator (Beck et al., Gene, 1-3: 35-58 (1981), and    -   the lad repressor gene from E. coli (Farabaugh, P. J., Nature,        274 (1978) 765-769).

The HC Plasmid 18023 is an expression plasmid for the expression of afusion protein including an antibody heavy chain, a linker containing afurin cleavage site, and a mutant of domain III of Pseudomonas ExotoxinA, in E. coli. It was generated by ligation of the VH domain fragmentinto the vector using the NdeI/BsrGI restriction sites. The specific PEvariant fragment was exchanged using the BsrGI/BsiWI restriction sites.

The cytolytic fusion protein heavy chain E. coli expression plasmidcomprises the following elements:

-   -   the origin of replication from the vector pBR322 for replication        in E. coli (corresponding to positions 2517-3160 according to        Sutcliffe et al., Quant. Biol., 43: 77-90 (1979),    -   the URA3 gene of Saccharomyces cerevisiae coding for orotidine        5′-phosphate decarboxylase (Rose et al., Gene, 29: 113-124        (1984), which allows plasmid selection by complementation of E.        coli pyrF mutant strains (uracil auxotrophy),    -   the heavy chain-PE domain III fusion protein expression        cassette, comprising:    -   the T5 hybrid promoter (T5-PN25/03/04 hybrid promoter according        to Bujard et al., Methods. Enzymol., 155: 416-433 (1987) and        Stueber et al., Immunol. Methods IV, 121-152 (1990), including a        synthetic ribosomal binding site according to Stueber et al.        (supra),    -   the antibody heavy chain variable domain comprising the CDRs of        SS1,    -   the human CH1 domain,    -   the linker comprising a furin cleavage site,    -   the mutant variant of Pseudomonas Exotoxin A domain III,    -   two bacteriophage-derived transcription terminators, the λ-T0        terminator (Schwarz et al., Nature, 272: 410-414 (1978) and the        fd-terminator (Beck et al., Gene, 1-3: 35-58 (1981), and    -   the lad repressor gene from E. coli (Farabaugh, Nature, 274:        765-769 (1978).

Expression of cFP-0205 in E. coli: The E. coli K12 strains CSPZ-6(thi-1, ApyrF) and CSPZ-13 (thi-1, ΔompT, ΔpyrF) were transformed byelectroporation with the expression plasmid nos. 15496 and 18023,respectively. The transformed E. coli cells were first grown at 37° C.on agar plates. For each transformation, a colony picked from this platewas transferred to a 3 mL roller culture and grown at 37° C. to anoptical density of 1-2 (measured at 578 nm). Then 1000 μl of culturewere mixed with 1000 μl sterile 86%-glycerol and immediately frozen at−80° C. for long term storage. The correct product expression of theseclones was first verified by small scale shake flask experiments andanalyzed with SDS-Page prior to the transfer to the 10L fermenter.

Pre Cultivation:

For pre-fermentation, a chemically defined medium (CD-PCMv2.60) wasused: NH₄Cl 1.25 g/l, K₂HPO₄*3H₂O 18.3 g/l, citrate 1.6 g/l,L-Methionine 0.20 g/l, Glucose*H₂O 8.0 g/l, trace elements solution 0.5ml/l, MgSO₄*7H₂O 0.86 g/l, and Thiamin*HCl 17.5 mg/l. The trace elementssolution contained FeSO₄*7H₂O 10.0 g/l, ZnSO₄*7H₂O 2.25 g/l, MnSO₄*H₂O2.13 g/l, H₃BO₃ 0.50 g/l, (NH₄)6Mo₇O₂₄*4H₂O 0.3 g/l, CoCl₂*6H₂O 0.42g/l, CuSO₄*5H₂O 1.0 g/l dissolved in 0.5M HCl.

For pre-fermentation, five 1000 mL shake-flasks with four baffles filledwith 220 mL of medium were inoculated with 1.0 mL each out of a primaryseed bank ampoule. The cultivation was performed on a rotary shaker for11-12 hours at 37° C. and 170 rpm until an optical density (578 nm) of 7to 9 was obtained. The shake flasks were pooled, and the optical densitywas determined. The inoculum volume was calculated with Vinoc.=1000mL*5/ODPC and is dependent on the optical density of the pre cultivationto inoculate the batch medium of each 100L bioreactor run with an equalamount of cells.

100L Scale Fermentation:

For fermentation in a 100L fermenter (Sartorius, Melsungen, Germany),the following chemically defined batch medium was used: KH₂PO₄ 1.59 g/l,(NH₄)₂HPO₄ 7.45 g/l, K₂HPO₄*3H₂O 13.32 g/l, citrate 2.07 g/l,L-methionine 1.22 g/l, NaHCO₃ 0.82 g/l, trace elements solution 7.3ml/l, MgSO₄*7H₂O 0.99 g/l, thiamine*HCl 20.9 mg/l, glucose*H₂O 29.3 g/l,biotin 0.2 mg/l, 1.2 ml/l Synperonic 10% anti foam agent. The traceelements solution contained FeSO₄*7H₂O 10 g/l, ZnSO₄*7H₂O 2.25 g/l,MnSO₄*H₂O 2.13 g/l, CuSO₄*5H₂O 1.0 g/l, CoCl₂*6H₂O 0.42 g/l,(NH₄)6Mo₇O₂₄*4H₂O 0.3 g/l, H₃BO₃ 0.50 g/l solubilized in a 0.5M HClsolution.

The feed 1 solution contained 700 g/l glucose*H₂O, 7.4 g/l MgSO₄*7 H₂Oand 0.1 g/l FeSO₄*7H₂O. Feed 2 comprised KH₂PO₄ 52.7 g/l, K₂HPO₄*3H₂O139.9 g/l and (NH₄)₂HPO₄ 66.0 g/l. All components were dissolved indeionized water. The alkaline solution for pH regulation was an aqueous12.5% (w/v) NH₃ solution supplemented with 11.25 g/l L-methionine.

Starting with 42L of sterile batch medium plus the calculated inoculumof the pre cultivation the batch, fermentation was performed at 31° C.,pH 6.9±0.2, 800 mbar back pressure and an initial aeration rate of 50l/min. The relative value of dissolved oxygen (pO²) was kept at 50%throughout the fermentation by increasing the stirrer speed up to 1000rpm. After the initially supplemented glucose was depleted, indicated bya steep increase in dissolved oxygen values, the temperature was shiftedto 37° C. when producing the light chain or kept constant when producingthe heavy chain construct and 15 minutes later, the fermentation enteredthe fed-batch mode with the start of both feeds (600 and 140 g/hrespectively). The rate of feed 2 was kept constant, while the rate offeed 1 was increased stepwise with a predefined feeding profile from 600to finally 1400 g/h when producing the light chain or 1600 g/h whenproducing the heavy chain construct within 6-7 hours. When carbondioxide off gas concentration leveled above 2%, the aeration rate wasstepwise increased by 10 L/min from 50 to 100 l/min within 5 hours. Theexpression of recombinant target proteins as insoluble inclusion bodieslocated in the cytoplasm was induced by the addition of 24 g IPTG at anoptical density of approx. 40 for the variable light Fab-chain and 120for the variable heavy Fab-chain.

After 24 hours of cultivation, the whole broth was cooled down to 4-8°C. and stored overnight in the fermenter vessel. The bacteria wereharvested via centrifugation with a flow-through centrifuge (13,000 rpm,13 l/h) or a separator and the obtained biomass was stored at −20° C.until further processing (cell disruption) or immediately processed forinclusion body isolation.

Analysis of Product Formation:

Samples were drawn from the fermenter, one prior to induction and theothers at dedicated time points after induction of protein expression.The samples were analyzed by SDS-Polyacrylamide gel electrophoresis. Forevery sample, the same amount of cells (OD_(Target)=10) was suspended in5 mL PBS buffer and disrupted via sonication on ice. Then, 100 μL ofeach suspension were centrifuged (15,000 rpm, 5 minutes) and eachsupernatant was withdrawn and transferred to a separate vial. This wasto discriminate between soluble and insoluble expressed target protein.To each supernatant (=soluble protein fraction) 100 μL and to eachpellet (=insoluble protein fraction) 200 μL of SDS sample buffer(Laemmli, Nature, 227: 680-685 (1970) were added. Samples were heatedfor 15 minutes at 95° C. under intense mixing to solubilize and reduceall proteins in the samples. After cooling to room temperature, 5 ofeach sample were transferred to a 4-20% TGX Criterion Stain Freepolyacrylamide gel (Bio-Rad). Additionally, 5 μL molecular weightstandard (Precision Plus Protein Standard, Bio-Rad) and 3 amounts (0.3μL, 0.6 μL and 0.9 μL) quantification standard with known target proteinconcentration (0.1 μg/μL) were applied.

The electrophoresis was run for 60 Minutes at 200 V and, thereafter, thegel was transferred the GELDOC EZ Imager (Bio-Rad) and processed for 5minutes with UV radiation. Gel images were analyzed using Image Labanalysis software (Bio-Rad). With the three standards, a linearregression curve was calculated with a coefficient of >0.99 and theconcentrations of target protein in the original sample were calculated.The yield was up to 3-4 g/L of the Fab-light chain and 10-12 g/L for theFab-heavy chain PE fusion.

Inclusion Body Preparation:

The inclusion body preparations (IBP) of the 100 L fermentations werestarted directly after the harvest of the bacteria with there-suspension of the harvested bacteria cells in buffer 1 (12.1 g/LTris, 0.246 g/L MgSO₄*7H₂O, 12 mL/L 25%-HCl). The buffer volume wascalculated in dependence of the dry matter content of the biomass.Lysozyme (100 kU/mg, 0.12 mg/gDCW) and a small amount of benzonase (5U/gDCW) were added. Then, the suspension was homogenized at 900 bar (APVRannie 5, 1 pass) to disrupt the bacteria cells followed by the additionfurther benzonase (30 U/gDCW) and an incubation for 30 minutes at 30-37°C. Then, the first wash buffer (60 g/L Brij, 87.6 g/L NaCl, 22.5 g/LEDTA, 6 mL/L 10N NaOH) was added and again the suspension was incubatedfor 30 minutes. The following separation step (CSC6, Westfalia) led toan inclusion body slurry which was re-suspended in the second washbuffer (12.1 g/L Tris, 7.4 g/L EDTA, 11 mL/L 25%-HCl) and incubated for20 minutes. A further separation step harvested the inclusion bodies toa single use, sterile, plastic bag, which was immediately transferred tothe DSP department.

Results:

The fermentations of the light chain yielded an optical density measuredat 578 nm of 170-190 and a final product yield of 10-12 g/L. Thefermentations of the heavy chain toxin fusion yielded an optical densitymeasured at 578 nm of 210-230 and a final product yield of 3-4.5 g/L.

Refolding and purification of cFP-0205: Inclusion bodies of HC-PE24 andLC were solubilized separately in 8 M guanidinium-hydrochloride, 100 mMTris/HCl, 1 mM EDTA, pH 8.0+100 mM dithiothreitol (DTT) overnight at RT(1 g IB in 5 mL). Solubilizates were adjusted to pH 3 and centrifuged,and the pellet was discarded. After extensive dialysis against 8 Mguanidinium-HCl, 10 mM EDTA, pH 3.0 to remove DTT, the total proteinconcentration was determined using the Biuret method. The purity of theHC and LC content was estimated via SDS-PAGE.

Solubilizates were diluted at a 1:1 molar ratio in renaturation buffercontaining 0.5 M arginine, 2 mM EDTA, pH 10+1 mM GSH/GSSG, respectively,at 2-10° C. The target protein concentration was increased stepwise from0.1 g/L up to 0.5 g/L, with an incubation time of 2 hours between eachdose. After up to 5 pulses, the renaturation solution was kept at 2-10°C. overnight.

The renaturate was diluted with H₂O to <3 mS/cm and pumped onto an anionexchange column (AIEX) equilibrated in 20 mM Tris/HCl, pH 7.4. Afterwashing the column with equilibration buffer, the protein was elutedwith a gradient up to 20 mM Tris/HCl, 400 mM NaCl, pH 7.4. Peakfractions containing Fab-PE24 were pooled, concentrated and applied ontoa preparative Size Exclusion Column (SEC) in 20 mM His, 140 mM NaCl, pH5.5 or 6.0 to remove aggregates, fragments, and E. coli proteins. Thefinal protein pool was adjusted to the required protein concentrationand analyzed via SDS-PAGE or CE-SDS, analytical SEC and UV₂₈₀. Identitywas confirmed by mass spectrometry.

Protein Analysis of cFP-0205: Sample analysis was carried out asdescribed above.

Characterization of Final Deimmunized PE Variant as Fab Fusion withHumanized Anti-Mesothelin SS1 (cFP0205).

Cytotoxicity and In Vitro Tumor Cell Killing:

The cytotoxic potency of cFP0205 was compared to that of SS1P in cellviability assays with different cancer cell lines and primarymesothelioma cells. The results are shown in Table 24. As shown in Table24, for many cancer cell lines, SS1P and cFP0205 had comparablepotencies (difference in IC₅₀ values <3). However, some cell lines(e.g., Hu-1 and Hu-2) and also the primary mesothelioma cells (RH19,RH21) were significantly more sensitive to cFP0205 than to SS1P (6-10fold lower IC₅₀ values).

TABLE 24 SS1P cFP0205 Cell line (IC50 in ng/ml) (IC50 in ng/ml) Hu-1 20.25 Hu-2 0.3 0.04 Hu-3 2.9 2 HAY 1 1.4 AG5 0.3 1.1 L55 3 5 MKN-28 0.5 2ASPC-1-luc 1.3 2.4 H596 4.6 15.7 RH19 3.7 1.2 RH21 2.3 0.35Characterization of Final Deimmunized PE Variant as Fab Fusion withHumanized Anti-Mesothelin SS1 (cFP0205).

Serum half life: A pilot study was performed in cynomolgus monkeys (1male and 1 female) with cFP0205 in order to assess the pharmacokineticsof single doses. The animals were intravenously dosed with 0.3 mg/kg ofcFP0205. For pharmacokinetics, blood samples were taken predose, 0.083(5 minutes, end of infusion), 1, 2, 3, 5, 8, 24, 48 and 168 hourspostdose. Levels of free drug (dashed line) as well as total drug (blacksquares and solid line) were determined by different ELISA formats (FIG.20). For determining total drug levels, a one-step acid dissociation wasperformed before the capturing step on mesothelin coated plates. Thefree and total drug levels were plotted over time. For comparison,historical data with SS1P were also inserted into FIG. 20 (black circlesand solid line). These historical data were generated with a cytotoxicactivity assay as a read-out for serum levels of SS1P. The cytotoxicityassay detects free drug, but also detects, at least in part, drug boundto soluble mesothelin which might contribute to the observed activity.The measured free drug levels for cFP0205 were similar to the firstphase of the historical data with SS1P. The measured total drug levelsfor cFP0205 were comparable to the second phase of the kinetic observedfor SS1P. In summary, not only in mice, but also in cynomolgus monkey,the serum level kinetics of SS1P and cFP0205 were very comparable.

Characterization of Final Deimmunized PE Variant as Fab Fusion withHumanized Anti Mesothelin SS1 (cFP0205)

Reduced Off-Target Toxicity:

SCID beige mice with small subcutaneous tumors of H596 lung cancer cells(average tumor volume 100 mm³) were dosed with 0.5, 1, 2, and 3 mg/kgcFP0205 3×/w qod and body weight loss was monitored over one month. Theresults are shown in FIG. 21.

As shown in FIG. 21, the maximally tolerated dose of SS1P in mice givenintravenously 3×/w qod was 0.4 mg/kg. cFP0205 given with this sameregimen was tolerated in SCID beige mice at doses up to 3 mg/kg. Somebody weight loss was observed in all treatment groups. However, theeffect of the 0.5 mg/kg dose of cFP0205 was indiscriminable from thevehicle control. Dose-dependently, an increasing loss of body weightoccurred with 1, 2, and 3 mg/kg. In the 3 mg/kg group, the maximumweight loss was 15% and this was observed 4 days after the lastapplication of cFP0205. In the 2 and 3 mg/kg treatment groups, nofurther decline in body weight was observed after the last application.All animals started to recover body weight from day 5 after the lasttreatment onwards. Initial body weight recovery was most pronounced inthe 3 mg/kg group. In summary, despite similar pharmacokineticproperties of both molecules in mice, cFP0205 was tolerated at an almost10 fold higher dose compared to SS1P.

A single intravenous dose (short infusion) toxicology study was done in8 week old female Wistar Furth rats (150-175 g body weight) in order toevaluate and compare the off-target toxicity of SS1P and cFP0205. Inparticular, the risk of inducing vascular leak (edema) in the lungcaused by the test item was investigated. Three animals per groupreceived the active substance or vehicle by an intravenous shortinfusion at an infusion rate of 0.3 ml/min. Animals were necropsied 24hours after dosing. Lung with mainstem bronchi and liver weremacroscopically evaluated, fluid collection in the lung was assessed,and clinical chemistry assays were performed on blood samples. Theresults are shown in Table 25.

TABLE 25 SS1P CFP0205 Dose (i.v.), n = 3 2 mg/kg 10 mg/kg In-life rales(lung), rolling gait, hunched posture, none (clinical piloerection andslight bw loss signs, bw) Necropsy tan-stained thoracic fluid in 2 rats;brown none discoloration of liver in all 3 rats Fluid Smear mesothelialcells and eosinophils none Evaluation Clinical Liver: ↑↑↑ in ALT (30x),AST (40x), Liver: Chemistry γGT, GLDH, SDH; marginal VLS: ↓ Protein, ↓albumin (serum SDH ↑ protein loss) Hemolysis?: ↑ bilirubin (but nohematology indication) ↑ BUN: poor physical condition (↑ proteincatabolism)The serum half life of SS1P and cFP0205 is expected to be very similarin rats as shown for mice.

As shown in Table 25, two out of three rats that were dosedintravenously with 2 mg/kg of SS1P showed fluid accumulation in thelungs. All animals in this group showed clear clinical signs of toxicityand, for all animals, clinical chemistry values were indicative ofsevere liver damage. In contrast to this, all three animals treated with10 mg/kg of cFP0205 showed no signs of toxicity. Only in one animal wasthe value for one of the liver enzymes slightly elevated. These findingsdemonstrate that the cFP0205 molecule causes much less hepatotoxicityand vascular leak syndrome than the SS1P classical immunotoxin format.

A pilot toxicology study was performed in cynomolgus monkeys (1 male and1 female) with cFP0205 in order to determine the tolerability ofrepeated daily doses of the test item following intravenousadministration and to check for potential late onset toxicity (up to 72hrs after last repeat-dose). Postdosing observations of the animals wereperformed immediately, 0.25, 0.5, 1, 2, and 4 hours after end of dosing.During the dosing phase, the general behavior and appearance of theanimals was observed twice daily, while body weight, food consumptionand feces were analysed daily.

Dosing cFP0205 at 1 mg/kg daily for 5 consecutive days was welltolerated by both animals. No hemorrhaging, acute inflammation, orulceration at the injection site were observed. That the animals showedno clinical signs of toxicity or non-tolerability firmly established 1mg/kg/d 5× as the non-severely toxic dose, while the classicalimmunotoxin format represented by SS1P had previously been shown to benon-tolerated at a dose of 0.3 mg/kg/d 5× with animals showingpersistent clinical signs of toxicity like hunched posture with tremors,hypoactivity, and poor appetite. In summary, cFP0205 is much bettertolerated than SS1P not only in mice, but also in cynomolgus monkeys, afully cross-reactive species for the targeting moieties of theseimmunoconjugates.

Characterization of Final Deimmunized PE Variant as Fab Fusion withHumanized SS1 (cFP0205)

cFP0205 and SS1P were labeled with Cy5 on free amino groups. OutstagedSCID beige mice with subcutaneous H596 tumors were intravenouslyinjected (4 mice/group) at a dose of 2 mg/kg with the 2fluorescent-labeled molecules. After 6 hours, the animals weresacricifed. The lung, liver, and spleen were formalin-fixed, embeddedand sectioned. Fluorescence images of representative sections of theseorgans were taken at different magnification. The results are shown inFIGS. 22A-22L.

As shown in FIGS. 22A-22L, SS1P treatment led to pronounced fluorescencestaining of lung and spleen tissue and massively stained what appearedto be the reticuloendothelial system in the liver. In contrast to this,there was no staining of lung tissue observed upon cFP0205 application.Also, staining of the spleen was much less pronounced and the stainingpattern was different from that of SS1P treated animals. Also in theliver, the staining with cFP0205 was drastically reduced compared toSS1P. Only isolated single cells that appear to be Kupffer cells werefluorescently stained. This suggests that the lower off-target toxicityof a Fab-PE24 compared to a dsFv-PE38 is due to differences in normaltissue distribution.

Example 15

This example demonstrates the production of anti-glypican 3-PE variantchimeric molecules.

An anti-glypican 3-PE variant chimeric molecule comprising a linkercomprising the amino acid sequence of SEQ ID NO: 36, a PE comprising SEQID NO: 37 (L010R-456A), the variable heavy chain domain VH of SEQ ID NO:61, and the variable light chain domain of SEQ ID NO: 62(GPC3-PE24-LR-LO10R-456A-long-linker) was prepared by a method analogousto the method of preparing the anti-mesothelin PE variant chimericmolecule cFP-077 described in Example 12 (from cloning to purification).

An anti-glypican 3-PE variant chimeric molecule comprising a linkercomprising the amino acid sequence of SEQ ID NO: 36, a PE comprising SEQID NO: 37 (L010R-456A), the variable heavy chain domain VH of SEQ ID NO:77, and the variable light chain domain of SEQ ID NO: 78 was alsoprepared by a method analogous to the method of preparing theanti-mesothelin PE variant chimeric molecule cFP-077 described inExample 12 (from cloning to purification).

Example 16

This example demonstrates the cytotoxic properties of the anti-glypican3-PE variant chimeric molecule GPC3-PE24-LR-LO10R-456A-long-linker ascompared to GPC3-PE24-LR-LO10R.

The cytotoxic potency of GPC3-PE24-LR-LO10R was compared to that ofGPC3-PE24-LR-LO10R-456A-long-linker on the glypican 3-positive livercancer cell line HepG2. Briefly, HepG2 cells were seeded at a density of6000 cells/well on 96 well plates. After overnight culture, differentconcentrations of the cFPs were added to the medium, and the cells wereincubated for 72 hours. At the end of incubation period, cell viabilitywas determined using a CELLTITERGLOW assay. The results are shown inTable 26.

As shown in Table 26, comparable dose-response curves for the inhibitionof cell viability were observed on HepG2 cells for both molecules. TheIC₅₀ value of the more completely de-immunized PE variant with the 456Amutation and the longer linker was even slightly lower than that of theLR-LO10R variant with the short linker. Non-targeted (“free PE24”) ormistargeted PE24 (“CD33-Fab-PE”; HepG2 cells do not express CD33) didnot show any significant reduction of cell viability at concentrations<30 μM. In summary, the more completely de-immunized GPC3-targetedLR-LO10R-456A-long-linker variant had the same or even slightly bettercytotoxic potency as compared to the GPC3-targeted LR-LO10R variant withthe shorter linker. Due to the additional 456A mutation within the PE,all B-cell epitopes were removed.

TABLE 26 IC50 in CELLTITERGLOW assay with HepG2 Fusion protein(expressing glypican-3) GPC3-PE24-LR-LO10R-short-linker 0.68 nMGPC3-PE24-LR-LO10R-456A-long-linker 0.54 nM Non-targeted PE24 >>30 μMCD33-PE24-LR-LO10R-short-linker no effect up to 30 μM

Example 17

This example demonstrates the cytotoxicity of deimmunized PE fusionproteins having a A458R mutation.

The cytotoxicity of SS1P, SS1-LR-GGS, SS1-LR-GGS-LO10,SS1-LR-GGS-LO10-A458R, and SS1-LR-GGS-LO10-A458-456A was measured usingthe WST method. The results are shown in Table 27.

TABLE 27 A431/H9 HAY M30 Hu-Meso AGS SS1P 0.05(N = 14) 1.49(N = 6)0.28(N = 5) 0.48(N = 5) 0.36(N = 5) SS1-LR-GGS 0.16(N = 2) ND ND ND 0.3SS1-LR-GGS-L010 0.17(N = 5) 0.68 3.99 0.11   11(N = 2)SS1-LR-GGS-L010-A458R 0.13(N = 18) 0.39(N = 11) 0.57(N = 3) 0.03(N = 4)1.05(N = 9) SS1-LR-GGS-L010-A458R- 0.07(N = 4)  0.1(N = 2) 0.58(N = 3)ND 0.5 456A

As shown in Table 27, the A458R mutation increased the activity ofSS1-LR-GGS-L010 on several cell lines. The addition of the 456A mutationfurther increased activity on several cell lines. A431/H9 is anepidermoid carcinoma expressing mesothelin, and Hay is a mesotheliomaline. M30 is also a mesothelioma cell line, and AGS is a stomach cancercell line. Hu-Meso are cells from a mesothelioma patient that wereplaced in culture for a few months.

Example 18

This example demonstrates the treatment of HCC70 tumors using RG7787.

Female nude mice were innoculated into the intramammary fat pad withHCC70 cells at time 0. HCC70 is a breast cancer cell line. Intravenoustreatment with RG7787 (2.5 mg/kg IV) or vehicle was begun on day 6 andcontinued every other day for a total of 5 doses (arrows). The size ofthe tumor was measured. The results are shown in FIG. 24.

As shown in FIG. 24, the tumor size of mice treated with RG7787 wasdecreased as compared to that of control mice (vehicle). There is astatistically significant difference between the 2 groups beginning atday 10, with p<0.00001 by day 14.

Example 19

This example demonstrates the treatment of KLM1 tumors in mice using acombination of paclitaxel and RG7787.

Four week, four day old mice were injected with 4×10⁶ KLM1 cells. KLM1is a pancreatic cancer cell line that expresses mesothelin. Thirteendays later, mice were untreated or treated with paclitaxel (50 mg/kg),RG7787 (2.5 mg/kg), or a combination of RG7787 (2.5 mg/kg) andpaclitaxel (50 mg/kg). Tumor size was measured for up to 89 days aftertreatment. The results are shown in FIG. 23.

As shown in FIG. 23, the tumor size of mice treated with the combinationof paclitaxel and RG7787 was decreased as compared to that of controlmice (untreated) or that of the mice treated with paclitaxel alone orRG7787 alone.

Example 20

This example demonstrates the treatment of HCC70 tumors in mice using acombination of paclitaxel and RG7787.

Female athymic nude mice were inoculated with HCC70 cells at time 0.Animals were treated with vehicle, RG7787 with IP vehicle injection,paclitaxel with IV vehicle injection, or paclitaxel and RG7787combination. Paclitaxel (50 mg/kg) was administered by IP injection ondays marked with long arrows. RG7787 (2.5 mg/kg) was administered IV ondays indicated by short arrows. Mean tumor volumes for n=5 vehicle andn=6 mice treated with RG7787, paclitaxel or the combination areindicated by the markers. The results are shown in FIG. 25.

As shown in FIG. 25, the tumor size of mice treated with the combinationof paclitaxel and RG7787 was decreased as compared to that of controlmice (vehicle) or that of the mice treated with paclitaxel alone orRG7787 alone.

Example 21

This example demonstrates the treatment of MKN-28 tumors in mice using acombination of paclitaxel and R205.

Athymic nude mice were inoculated with MKN-28 cells at time 0. MKN-28 isa gastric cancer cell line which expresses mesothelin. Animals wereuntreated (UT) or treated with Roche 205 (R205 or 205) alone, paclitaxel(taxol) alone, or a combination of R205 and paclitaxel. Tumor size wasmeasured. The results are shown in FIG. 26.

As shown in FIG. 26, the tumor size of mice treated with the combinationof paclitaxel and R205 was decreased as compared to that of control mice(untreated) or that of the mice treated with paclitaxel alone or R205alone.

Example 22

This example demonstrates the in vivo antitumor activity of LMB-T18.

Severe combined immunodeficient (SCID) mice were implanted with CA46cells. Seven days later, when tumors reached over 100 mm³ in size, themice were treated with PBS (control) or LMB-T18 (5 mg/kg x 4 or 7.5mg/kg×3) intravenously. Mice receiving 5.0 mg/kg were treated fourtimes, on days 7, 9, 11 and 16, and the higher dose group was treatedwith 7.5 mg/kg three times on days 7, 9 and 11. Marked tumor regressionswere observed in all mice (FIG. 27E) with only minor weight loss(average of 6%). 5/7 mice treated with 5.0 mg/kg maintained completetumor regression on day 33, and 3/7 maintained complete tumor regressionin the 7.5 mg/kg group. To assess the nonspecific toxicity of LMB-T18,six tumor bearing mice were treated intravenously with two doses of 10mg/kg QOD. One mouse showed severe weight loss and was euthanized.

Example 23

This example demonstrates that LMB-T18 has greatly diminished T cellactivation.

To determine if LMB-T18 had a decrease in T cell stimulation or if new Tcell epitopes were created by the mutations, PBMCs were stimulated fromthe highest responder donors (n=13) and HCL and mesothelioma patients(n=7) with MP or LMB-T18. Cells were re-stimulated with the 39 novelpeptides representing the differences between MP and LMB-T18. A decreaseof 90% in donor T cell activation (p<0.0001 in Student T test) wasobserved. Even in patients with activated T cells, there was an 83%decrease. (p<0.0001 in Student T test). Furthermore, no new epitopeswere created by the mutations.

Example 24

This example demonstrates that LMB-T18 has reduced binding to anti-serafrom patients.

The antigenicity of LMB-T18 was evaluated by comparing the reactivity ofMP, HA22-LR and LMB-T18 with serum from patients with neutralizingantibodies to MP. Binding was measured using ICC-ELISA with serum from13 MP treated patients and is shown in FIG. 27F. It was found that likeHA22-LR, LMB-T18 had a significantly reduced binding to serum comparedto MP (p<0.001 one-way ANOVA). It was also found that LMB-T18 hadsignificantly lower binding compared to HA22-LR (p<0.001 one-way ANOVA),indicating that the mutations in LMB-T18 reduced the binding toanti-sera.

Example 25

This example demonstrates the production of anti-FAP-PE variant chimericmolecules.

Three anti-FAP-PE variant chimeric molecules comprising:

-   -   (a) a linker comprising the amino acid sequence of SEQ ID NO:        36, a PE comprising SEQ ID NO: 37 (L010R-456A)), the variable        heavy chain domain VH of SEQ ID NO: 93, and the variable light        chain domain of SEQ ID NO: 94        (iFAP-PE24-LR-LO10R-456A-long-linker)    -   (b) a linker comprising the amino acid sequence of SEQ ID NO:        36, a PE comprising SEQ ID NO: 37 ((L010R-456A)), the variable        heavy chain domain VH of SEQ ID NO: 93, and the variable light        chain domain of SEQ ID NO: 294        (LCL4-PE24-LR-LO10R-456A-long-linker) (including the VL variant        of SEQ ID NO: 290), and    -   (c) a linker comprising the amino acid sequence of SEQ ID NO:        36, a PE comprising SEQ ID NO: 37 (T-20), the variable heavy        chain domain VH of SEQ ID NO: 93, and the variable light chain        domain of SEQ ID NO: 290 (LCL4-T20);

were prepared by a method analogous to the method of preparing theanti-mesothelin PE variant chimeric molecule cFP-077 described inExample 12 (from cloning to purification), except as described below.

The anti-FAP-PE variant chimeric molecules (b) and (c) included amodified variable light chain domain (VL) in which the methionine atposition 4 of the original VL sequence of SEQ ID NO: 94 was substitutedwith leucine to provide a modified VL sequence of SEQ ID NO: 290(“LCL4”). The modified variable light chain domain SEQ ID NO: 290 wasfound to reduce the production of impurities related to N-terminalmodifications. These N-terminal modifications provided a mixture ofvariants with different light chain lengths due to the initiation ofexpression of the unmodified sequence at either the N-terminalmethionine or the methionine at position 4 of of SEQ ID NO: 94.

Pre-Cultivation:

For pre-fermentation, the same chemically defined medium (CD-PCMv2.20)was used as described in Example 12.

The cultivation was performed on a rotary shaker for 8 hours at 37° C.and 170 rpm until an optical density (578 nm) of 13.3 or 14.4 wasobtained. 150 ml of the pre-cultivation was used to inoculate the batchmedium of the 10L bioreactor.

Fermentation:

For fermentation in a 101 Biostat C, DCU3 fermenter (Sartorius,Melsungen, Germany), the same chemically-defined batch medium asdescribed in Example 12 was used.

One feed solution that was used contained 700 g/l glucose*H₂O, 7.4 g/lMgSO₄*7 H₂O and 0.1 g/l FeSO₄*7H₂O. All components were dissolved indeionized water. The alkaline solution for pH regulation was an aqueous12.5% (w/v) NH₃ solution supplemented with 11.25 g/l L-methionine and 10g/L Leucine and 10 g/L Threonine.

Starting with 6.24 L sterile batch medium plus 150 mL inoculum from thepre-cultivation, the batch fermentation was performed at 32° C., pH6.9±0.2, 800 mbar back pressure and an initial aeration rate of 10l/min. The relative value of dissolved oxygen (pO₂) was kept at 50%throughout the fermentation by increasing the stirrer speed up to 1500rpm. After the initially supplemented glucose was depleted, indicated bya steep increase in dissolved oxygen values, the temperature was shiftedto 32° C. or 37° C., and 15 minutes later, the fermentation entered thefed-batch mode with the start of both the feeds (60 and 14 g/hrespectively). The rate of the feed was increased stepwise with apredefined feeding profile from 90 to finally 210 or 240 g/h within 5.5or 6.5 hours. When carbon dioxide off gas concentration leveled above2%, the aeration rate was constantly increased from 10 to 20 l/minwithin 5 hours. The expression of recombinant protein was induced by theaddition of 3.6 g IPTG at an optical density of approx. 40 or 120. Thetarget protein was expressed as inclusion bodies within the cytoplasm.

After 24 hours of cultivation, an optical density of 179 or 171 wasachieved, and the whole broth was cooled down to 4-8° C. The bacteriawere harvested via centrifugation with lab centrifuge (4500 rpm, coolingat 4° C., for 1 h) and the obtained biomass was stored at −20° C. untilfurther processing (cell disruption).

Analysis of Product Formation:

The specific differences in comparison to the method of Example 12 aredescribed below:

The same amount of cells (OD_(Target)=10) from every sample weresuspended in 5 mL PBS buffer and disrupted via sonication on ice. Then100 μL of each suspension were centrifuged (8,000 rpm, 5 minutes) andeach supernatant was withdrawn and transferred to a separate vial.

After adding Laemmli buffer, the samples were heated for 45 minutes 40°C. under intense mixing to solubilize and reduce all proteins in thesamples.

Inclusion Body Preparation:

The inclusion body preparations (IBP) of the 10L fermentations wereprocessed in a manner analogous to the method described in Example 12,with the exception that after disruption of the bacteria cells, furtherbenzoase (30 U/gDCW) was added and incubated for 60 minutes at 25° C.

Solubilization, renaturation and purification were performed in a mannersimilar to that described in Example 12. The following sequences wereused:

-   -   Sequence of iFAP-PE24-LR-LO10R-456A-long-linker and        LCL4-PE24-LR-LO10R-456A-long-linker chimeric full length heavy        chain comprising the variable domain VH of SEQ ID NO: 93, a        linker comprising the amino acid sequence of SEQ ID NO: 36, and        a PE comprising SEQ ID NO: 37 (SEQ ID NO: 291);    -   Sequence LCL4-T20 chimeric heavy chain construct comprising the        full length heavy chain comprising the variable domain VH of SEQ        ID NO: 93, a linker comprising the amino acid sequence of SEQ ID        NO: 36 and a PE comprising SEQ ID NO: 289 (T18/T20) (SEQ ID NO:        292);    -   Sequence of full length iFAP light chain (SEQ ID NO: 293)        comprising the variable light chain domain of SEQ ID NO: 94 and        constant region;    -   Sequence of full length LCL4 light chain (SEQ ID NO: 294)        comprising the variable light chain domain of SEQ ID NO: 290 and        constant region; and    -   Sequence of the variable light chain domain of SEQ ID NO: 290        (with mutation M4L at position 4 (compared to SEQ ID NO: 94).

Example 26

This example demonstrates the cytotoxicity of the anti-FAP-PE variantchimeric molecules iFAP-PE24-LR-LO10R-456A-long-linker,LCL4-PE24-LR-LO10R-456A-long-linker, and LCL4-T20.

The cytotoxicity of iFAP-PE24-LR-LO10R-456A-long-linker was compared tothat of LCL4-PE24-LR-LO10R-456A-long-linker and LCL4-T20 (“cFPs”) withrespect to the FAP-positive fibroblast cell line MRC-5, the FAP-positivedesmoplastic melanoma cell line LOX-IMVI, and the FAP-positive sarcomacell line OsA-CL. Briefly, cells were seeded at a density of 10,000cells/well on 96-well plates. After overnight culture, differentconcentrations of the cFPs were added to the medium, and the cells wereincubated for 72 hours. At the end of incubation period, cell viabilitywas determined using a CELLTITER GLO assay. The results are shown inTable 28 and Table 29.

As shown in Table 28, comparable IC50 values for the inhibition of cellviability were observed on all 3 tested cell lines foriFAP-PE24-LR-LO10R-456A-long-linker (3 different batches) and forLCL4-PE24-LR-LO10R-456A-long-linker. As shown in Table 29, LCL4-T20 alsoshowed potent inhibition of cell viability. In summary,iFAP-PE24-LR-LO10R-456A-long-linker,LCL4-PE24-LR-LO10R-456A-long-linker, and LCL4-T20 showed potentcytotoxic effects on FAP-positive tumor cell lines and fibroblasts.

TABLE 28 IC50 in CELLTITER GLO assay with cell lines expressingFAP(ng/ml) MRC-5 MRC-5 (experi- (experi- Fusion protein ment 1) ment 2)LOX-IMVI OsA-CL iFAP- PE24- 14.3 8.5 6.6 98.3 LR-LO10R- 456A-long-linker (Batch 1) iFAP- PE24- 14.4 9.3 9.1 46.3 LR-LO10R- 456A-long-linker (Batch 2) iFAP- PE24- 10.0 5.5 5.7 55.3 LR-LO10R- 456A-long-linker (Batch 3) LCL4- PE24- 13.9 9.8 9.7 103.1 LR-LO10R- 456A-long-linker

TABLE 29 IC50 in CELLTITER GLO assay with cell lines expressing FAP(nM)Fusion protein MRC-5 LOX-IMVI OsA-CL LCL4- PE24-LR-LO10R- 0.15 nM 0.33nM 0.23 nM 456A-long-linker LCL4-T20 0.98 nM 8.99 nM 0.50 nM

Example 27

This example demonstrates the production of anti-CAIX-PE variantchimeric molecules.

Two anti-CAIX-PE variant chimeric molecules comprising:

-   -   (a) a linker comprising the amino acid sequence of SEQ ID NO:        36, a PE comprising SEQ ID NO: 37 (L010R-456A), the variable        heavy chain domain VH of SEQ ID NO: 125, and the variable light        chain domain of SEQ ID NO: 126; or    -   (b) a linker comprising the amino acid sequence of SEQ ID NO:        36, a PE comprising SEQ ID NO: 289 (T-20), the variable heavy        chain domain VH of SEQ ID NO: 125, and the variable light chain        domain of SEQ ID NO: 126;        -   were prepared by a method analogous to that described for            the anti-mesothelin PE variant chimeric molecule cFP-077 of            Example 12 (from cloning to purification), except as            described below.

Samples were analyzed by OD 280 nm using a UV spectrophotometer todetermine the protein concentration in solution. The materials for theSDS-PAGE and Coomassie Staining Device are set forth in Table 30 below.

TABLE 30 Invitrogen XCell Sure Lock Mini-Cell Gel: 4-12% Bis-Tris Gel,Invitrogen NP0321 Buffer: MES SDS Running Buffer (10x), InvitrogenNP0002 Sample buffer: Tris-Glycine SDS Sample Buffer (2x), InvitrogenLC2676 Reducing buffer: NUPAGE Sample Reducing Agent (10x), InvitrogenNP0004 Molecular Precision Plus KALEIDOSCOPE Weight Marker: Standard161-0375

After adding Laemmli buffer, the samples were heated for 45 minutes at40° C. under intense mixing to solubilize and reduce all proteins in thesamples.

The sample was adjusted to a protein concentration of 1 mg/ml withbuffer.

Sample reduction used a reduction buffer comprising 4 ml of samplebuffer (2×) and 1 ml of reducing buffer (10×). The samples were reducedby diluting the sample 1:1 with reduction buffer and incubating thesample for 10 minutes at 70° C.

The gel electrophoresis was carried out at 200 V for 40 minutes. Thegels were stained with SIMPLY BLUE Safe Stain (Invitrogen, Cat. No.LC6065).

Inclusion Body Preparation:

The inclusion body preparations (IBP) of the 10L fermentations wereprocessed in a manner analogous to that described in Example 12, withthe exception that after disruption of the bacteria cells, furtherbenzoase (30 U/gDCW) was added and incubated for 60 minutes at 25° C.

Solubilization, renaturation and purification were performed in a mannersimilar to that described in Example 12.

The following sequences were used:

-   -   Sequence of CAIX-PE24-LR-LO10R-456A-long-linker chimeric heavy        chain construct (SEQ ID NO: 295): comprising the variable heavy        chain domain VH of SEQ ID NO: 125, a linker comprising the amino        acid sequence of SEQ ID NO: 36, and a PE comprising SEQ ID NO:        37;    -   Sequence of CAIX-T20 chimeric heavy chain construct (SEQ ID        NO: 296) comprising the variable heavy chain domain VH of SEQ ID        NO: 125, a linker comprising the amino acid sequence of SEQ ID        NO: 36 and a PE comprising SEQ ID NO: 289 (T18/T20); and    -   Sequence of full length light chain (SEQ ID NO: 297) comprising        the variable light chain domain of SEQ ID NO: 126) and constant        region.

Example 28

This example demonstrates the cytotoxicity (in vitro tumor cell killing)of the anti-CAIX-PE variant chimeric moleculesCAIX-PE24-LR-LO10R-456A-long-linker and CAIX-T20.

The cytotoxicity of CAIX-PE24-LR-LO10R-456A-long-linker and CAIX-T20(“cFPs”) was compared in cell viability assays using CAIX-expressingRCC-MF cells. Briefly, cells were seeded at a density of 7,500cells/well on 96 well plates. After overnight culture, differentconcentrations of the cFPs were added to the medium, and the cells wereincubated for 72 hours. At the end of incubation period, cell viabilitywas determined using a CELLTITER GLO assay. The results are shown inTable 31.

In summary, CAIX-PE24-LR-LO10R-456A-long-linker and CAIX-T20 showedpotent cytotoxic effects on CAIX-positive tumor cell lines.

TABLE 31 IC50 in CELLTITER GLO assay with cell lines expressing CAIX(nM) Fusion protein RCC-MF cells CAIX-PE24-LR-LO10R-  0.02 nM456A-long-linker CAIX-T20 0.048 nM

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and “at least one” andsimilar referents in the context of describing the invention (especiallyin the context of the following claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The use of the term “at least one”followed by a list of one or more items (for example, “at least one of Aand B”) is to be construed to mean one item selected from the listeditems (A or B) or any combination of two or more of the listed items (Aand B), unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1-42. (canceled)
 43. A humanized anti-mesothelin antibody or antigenbinding fragment thereof comprising a variable heavy chain domainsequence that is SEQ ID NO: 45 and a variable light chain domainsequence that is SEQ ID NO:
 46. 44. An isolated nucleic acid encodingthe antibody or antigen binding fragment thereof of claim
 43. 45. Anexpression vector comprising the nucleic acid of claim
 44. 46. A hostcell comprising the expression vector of claim
 45. 47. A method forproducing a humanized anti-mesothelin antibody or antigen bindingfragment thereof comprising: (i) culturing the host cell of claim 46 ina medium; and (ii) purifying the antibody or fragment thereof from thecultured host cells.
 48. A composition comprising the antibody orantigen binding fragment of claim 43 and a carrier.
 49. A chimericmolecule comprising the antibody or antigen binding fragment of claim 43conjugated or fused to a Pseudomonas exotoxin A.